Aluminum compounds for use in therapeutics and vaccines

ABSTRACT

The invention relates to means and methods for preparing aqueous composition comprising aluminium and a protein said composition comprising less than 700 ppm heavy metal on the basis of weight with respect to the aluminium content. The invention further relates to aqueous compositions comprising a protein and an aluminium-salt, said composition comprising less than 350 ppb heavy metal based on the weight of the aqueous composition.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/873,344, filed Oct. 2, 2015, which is a divisional of U.S.application Ser. No. 13/449,596, filed Apr. 18, 2012, the disclosure ofeach of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to the fields of pharmaceuticals and vaccines.More in particular the invention relates to the field of compounds andcompositions that are being co-administered with the medicament and/orantigen.

BACKGROUND OF THE INVENTION

Aluminium compounds (herein also referred to as “aluminium”), includingaluminium phosphate (AlPO4), aluminium hydroxide (Al(OH)3), and otheraluminium precipitated vaccines are currently the most commonly usedadjuvants with human and veterinary vaccines. The adjuvants are oftenreferred to as “alum” in the literature.

Aluminium adjuvants have been used in practical vaccination for morethan half a century. They induce early, high-titre, long-lastingprotective immunity. Billions of doses of aluminium-adjuvated vaccineshave been administered over the years. Their safety and efficacy havemade them the most popular adjuvants in vaccines to date. In general,aluminium adjuvants are regarded as safe when used in accordance withcurrent vaccination schedules.

In human vaccinations, of old, aluminium adjuvants have been used intetanus, diphtheria, pertussis and poliomyelitis vaccines as part ofstandard child vaccination programmes. Aluminium adjuvants have alsobeen introduced into hepatitis A and hepatitis B virus vaccines andJapanese encephalitis virus (also referred herein as “JEV”) vaccines.Other aluminium-adsorbed vaccines against, for example, anthrax, areavailable for special risk groups. In veterinary medicine aluminiumadjuvants have been used in a large number of vaccine formulationsagainst viral and bacterial diseases, and in attempts to makeantiparasite vaccines.

Adjuvants typically serve to bring the antigen, the substance thatstimulates the specific protective immune response, into contact withthe immune system and influence the type of immunity produced, as wellas the quality of the immune response (magnitude or duration); Adjuvantscan also decrease the toxicity of certain antigens; and providesolubility to some vaccines components. Studies have shown that manyaluminium-containing vaccines cause higher and more prolonged antibodyresponses than comparable vaccines without the adjuvant. The benefit ofadjuvants has usually been observed during the initial immunizationseries rather than with booster doses.

There are three general types of aluminium-containing adjuvants:

Aluminium hydroxide, Aluminium phosphate and Potassium aluminiumsulphate (collectively often referred to as “Alum”)

The effectiveness of each salt as an adjuvant depends on thecharacteristics of the specific vaccine and how the manufacturerprepares the vaccine. To work as an adjuvant, the antigen is typicallyadsorbed to the aluminium; that is, it is clumped with the aluminiumsalt to keep the antigen at the site of injection.

Not all vaccines contain aluminium salts. Sometimes an adjuvant may nothave been needed or another adjuvant was selected. Examples ofcommercial vaccines that do not contain aluminium salts are inactivatedPolio Virus (IPV) vaccine, measles, mumps and rubella vaccine (MMR),varicella vaccine, Meningococcal conjugate (MCV4) vaccine, and influenzavaccines. That the commercial vaccines do not contain aluminium saltsdoes typically not mean that an aluminium salt would not work. It justmeans that for some reason another adjuvant was selected.

Examples of US licensed vaccines for children that contain aluminiumadjuvants are: DTP (diphtheria-tetanus-pertussis vaccine); DTaP(diphtheria-tetanus-acellular pertussis vaccine); some but not all Hib(Haemophilus influenzae type b) conjugate vaccines; Pneumococcalconjugate vaccine; Hepatitis B vaccines; Hepatitis A vaccines; HumanPapillomavirus vaccine; Anthrax vaccine; and Rabies vaccine.

Aluminium is a very abundant element in our environment. It is in manyfoods we eat, many personal hygiene products we apply to our skin(deodorants, for example), and many medicines we ingest. Variousgovernment agencies establish guidelines for exposure to potentiallytoxic substances. These guidelines are called “minimal risk levels”—themaximum amount that one can be exposed to over time-usually on a dailybasis-without expected harm.

The US Agency for Toxic Substances and Disease Registry (ATSDR)estimated these levels for infants taking into account the amount ofaluminium (e.g. in form of a salt) a child would eat as well as receiveby injection of vaccines. The body burden of aluminium from both sourcesis below the minimal risk level except transiently followingvaccinations; since 50-70% of injected aluminium is excreted within 24hours, this is believed to have no negative effect.

Aluminium hydroxide and aluminium phosphate adjuvants are generallyprepared by exposing aqueous solutions of aluminium ions, to usuallyslightly alkaline conditions in a well-defined and controlled chemicalenvironment. Various soluble aluminium salts can be used for theproduction of aluminium hydroxide. Anions present at the time ofprecipitation may coprecipitate (for review see, Lindblad, E B (2004)Immunol. and Cell Biol. Vol 82: 497-505).

Aluminium salt is also used in the manufacture and composition ofmedicaments. For instance, factor VIII is purified from plasmacryoprecipitate. The precipitate is solubilised, absorbed on aluminiumhydroxide and then treated to inactivate lipid enveloped viruses. Afterseveral other processing steps the concentrate is used to treathemophilia A patients (Burnouf T, (1991) Vox Sang. Vol 60: pp 8-15).

SUMMARY OF THE INVENTION

In the present invention it has been shown that stability of abiological in a composition that also comprises an aluminium salt is notalways the same. The present invention, for instance, shows that thestability of a protein component (e.g. as such or within a complex suchas e.g. a virus or other pathogen) in the context of an aqueouscomposition that also comprises aluminium salt is dependent on thecontent of heavy metals. To estimate a priori whether the protein willbe stable in this composition, the present invention provides that it isnecessary to determine the residual heavy metal content in thecomposition (otherwise the aqueous composition comprising a protein isat risk of being degraded over time in particular the invention providesthat this risk is considerable when the residual heavy metal content isabove 350 ppb (i.e. about 350 ng per ml) in said aqueous composition).Further, the invention also revealed that this residual heavy metalcontent cannot easily be removed from the aluminium compound. To thisend the invention provides a method for preparing an aqueous compositioncomprising aluminium and a protein said method comprising—combining analuminium-salt, said protein and water to produce said aqueouscomposition and—determining the level of a heavy metal in the aqueouscomposition and/or the aluminium-salt. Compositions comprising less than350 ppb heavy metal based on the weight of the aqueous composition, canbe stored in a liquid phase at a temperature of between 0 and 30 degreesCelsius, for at least 1 month, such as e.g. 20 months at 2-8° C. Theprotein component in said composition is stable for at least 1 month insaid liquid phase. Compositions comprising more than 350 ppb heavy metalbased on the weight of the aqueous composition cannot be stored for aprolonged period under such conditions as the protein component in saidcomposition changes in at least one aspect over the indicated timeperiod. One millilitre or one gram of aqueous composition thuspreferably contains no more than 350 nanogram heavy metal. The aqueouscomposition preferably comprises between 0.1 mg/ml and 2.5 mg/mlaluminium. The average dose of aluminium per administration ispreferably not more than 1.25 milligram (mgram). In a particularlypreferred embodiment the dose of aluminium per administration is notmore than 0.25 mgram aluminium. A dose typically comprises between 0.5and 1 ml of the aqueous composition.

In a further aspect of the present invention it has been shown thatstability of a biological in a composition that comprises an aluminiumsalt and a reactive compound is not always the same. The presentinvention, for instance, shows that the stability of a protein component(e.g. as such or within a complex such as e.g. a virus or otherpathogen) in the context of an aqueous composition that also comprisesaluminium salt and a reactive compound such as e.g. a sulphite iscritically dependent on the content of heavy metals. To estimate apriori whether the protein component (such as e.g. protein componentwithin a complex such as e.g. a virus particle; herein also referred tosimply as protein) will be stable in this composition, it is necessaryto determine the heavy metal content in the composition. To this end theinvention provides a method for preparing an aqueous compositioncomprising aluminium and a protein said method comprising—combining analuminium-salt, said protein and water to produce said aqueouscomposition and—determining the level of a heavy metal in the aqueouscomposition and/or the aluminium-salt. Compositions comprising less than350 ppb heavy metal based on the weight of the aqueous composition, canbe stored in a liquid phase at a temperature of between 0 and 30 degreesCelsius, for at least 1 month, such as e.g. 20 months at 2-8° C. Theprotein component in said composition is stable for at least 1 month insaid liquid phase, such as e.g. 20 months at 2-8° C. Compositionscomprising more than 350 ppb heavy metal based on the weight of theaqueous composition cannot be stored for a prolonged period under suchconditions as the protein component in said composition changes in atleast one aspect over the indicated time period. One millilitre or onegram of aqueous composition thus preferably contains no more than 350nanogram heavy metal. The aqueous composition preferably comprisesbetween 0.1 mg/ml (milligram per millilitre) and 2.5 mg/ml aluminium.The average dose of aluminium per administration is preferably not morethan 1.25 milligram (mgram). In a particularly preferred embodiment thedose of aluminium per administration is not more than 0.25 mgramaluminium. A dose typically comprises between 0.5 and 1 ml of theaqueous composition. An aqueous composition comprising a protein, analuminium-salt, and optionally a reactive compound, said compositioncomprising less than 350 ppb heavy metal based on the weight of theaqueous composition is herein also referred to as “an aqueouscomposition comprising a protein according to the invention” or “acomposition comprising a protein according to the invention”.

It has been observed that the aluminium component is an important sourcefor the heavy metal in the aqueous composition. Thus one way to controlthe amount of heavy metal in the aqueous composition is to control theamount of heavy metal in the aluminium source used to generate theaqueous composition. The invention therefore further provides a methodfor preparing an aqueous composition comprising aluminium and a proteinsaid method comprising

-   -   preparing or selecting an aluminium-salt solution (such as e.g.        10 mg/ml aluminium hydroxide liquid (such as e.g. ALHYDROGEL® 2%        from Brenntag Biosector, catalogue number 843261) that in the        final protein formulation comprises no more than 350 ppb based        on the weight of the aqueous composition (e.g. for the        ALHYDROGEL® 2% and final amount of 0.25 mgram aluminium        hydroxide in the aqueous composition of 0.5 ml (=dose), the        selected or prepared aluminium-salt solution should not contain        more than 7 microgram heavy metal pro millilitre of the        ALHYDROGEL® 2% solution, about 7 ppm heavy metal content), and    -   combining said aluminium-salt solution, said protein, water and        optionally a reactive compound to produce said aqueous        composition (with no more than 350 ppb heavy metal in the        aqueous composition).

For example, the aluminium-salt solution, e.g. the aluminium hydroxideliquid (used as a component to be mixed to result in the final aqueouscomposition) should not have a heavy metal content higher than 7 ppm(given as an example herein where the 10 mg/ml aluminium hydroxideliquid will be diluted to 0.5 mg/ml to result in about 350 ppb heavymetal content, assuming 1 ppm=about 1 mg/ml) on the basis of weight ofthe aluminium hydroxide liquid. The limit of acceptable heavy metalcontent may also be expressed in relation to the weight of thealuminium-salt such as the aluminium hydroxide in solution (referred toalso as “starting aluminium compound”). The acceptable heavy metalcontent in this example then may not exceed 7 microgram of heavy metalfor each gram of aluminium hydroxide solution (i.e. about 7 ppm), i.e.the ALHYDROGEL® 2% solution. As indicated herein above the aqueouscomposition comprising the protein preferably (such as e.g. if used as avaccine) comprises between 0.1 to 2.5 mg/ml of the aluminium compound.The concentration of heavy metal in the aqueous composition howevershould according to the invention not exceed 350 ppb, i.e. about 350 ngper ml of the final composition and thus the selection or preparation ofthe starting aluminium compound has to be made accordingly. In order tofurther illustrate the selection of an appropriate starting aluminiumcompound solution (e.g. in the form of a concentrated solution (seeabove ALHYDROGEL® 2%=10 mg/ml), it is shown that an aluminium compoundsolution of 10 mg/ml aluminium hydroxide having about 7 ppm heavy metalimpurities corresponds to a concentration of heavy metal in the proteincomposition when the aluminium concentration is about 0.1 mg/ml of about70 nanogram/ml or 70 ppb (so well below the 350 ppb provided as thelimit of the heavy metal content as taught by the invention). Aconcentration of 2.5 mg/ml of the aluminium hydroxide in the finalaqueous composition starting with an aluminium solution of 10 mg/mlaluminium hydroxide having about 7 ppm heavy metal impurities willresult in a heavy metal content in the aqueous protein composition thatcorresponds to a concentration of heavy metal of about 1.75 microgram/mlor about 1,750 ppm (so well above the 350 ppb provided as the limit ofheavy metal content as taught by the invention).

A method of preparing an aqueous composition comprising a proteinpreferably further comprises packaging aliquots of said aqueouscomposition having less than 350 ppb heavy metal based on the weight ofthe aqueous composition in separate air-tight storage containers. Theprotein in the air-tight storage containers is stable and can be storedfor at least three months, such as e.g. 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, preferably 20 or24 months, more preferably 20 months at a temperature of between 2 to 8°C. degrees Celsius.

Without being bound to theory, antigen degradation in aqueouscompositions, such as immunogenic composition, comprising heavy metalions present in an aluminium salt, such as aluminium hydroxide, might beexplained with an underlying degradation pathway assuming free-radicalssuch as e.g. free-radicals of sulphite. Formation of free radicals canbe catalysed by heavy metal ions present in, for instance, aluminiumhydroxide and this effect (in case of a certain amount of heavy metal asindicated according to the invention) could be the underlying root causemechanism for stability issues as identified as part of the inventivecontribution. The experimental part of this application shows in greatdetail the evidence of this root cause for the Japanese encephalitisvaccine (also referred to as “JEV”) and makes a similar showing for asimple aluminium adjuvanted polypeptide composition that comprises areactive compound such as sulphite. Thus, it is evident that similarreaction may occur also in other aqueous composition comprisingaluminium (with high, e.g. higher than 350 ppb based on the weight ofthe composition, heavy metal content), protein and possibly a reactivecomponent such as sulphite and/or other radical forming particles. Heavymetal-catalysed oxidation is a degradation pathway resulting in covalentmodification of proteins. The modified physicochemical properties of theoxidized/modified protein or antigen may result in loss of biologicalactivity (Li et al., 1995; Mayo et al., 2003; Stadtman, 1990).

The following reaction schemes (as possibly occurring in the JEV productas described in the experimental part) are indicative for compositionsof the invention comprising in addition to the protein and the heavymetal also sulphite such as e.g. sulphite and formaldehyde.

Sodium-metabisulphite (Na₂S₂O₅) in solution dissolutes into bisulphite(S₂O₅ ²⁻), in an alkaline solution the bisulphite equilibrium is towardssulphite (SO₃ ²⁻) and in an acidic solution towards H₂SO₃/SO₂. Afterdissolution of Na₂S₂O₅ in water, it is hydrolysed into NaHSO₃ as follows

Na₂S₂O₅

2NaHSO₃

At neutral pH we can assume the following equilibrium:

HSO³⁻

H⁺+SO₃ ²⁻pKa=7.2

This means that at pH 7 the equilibrium is shifted towards HSO3- and atmore basic conditions (e.g. pH 8) towards SO₃ ²⁻.

Formaldehyde forms a bisulphite adduct during neutralization accordingto the following equation:

CH₂O+HSO³⁻→CH₂(OH)(SO₃)⁻

After bisulphate is used up, the reaction proceeds until equilibrium isreached as follows:

CH₂O+SO₃ ²⁻+H₂O→CH₂(OH)(SO₃)⁻+OH⁻

Formaldehyde and sulphite react with each other, however, formaldehydeand sulphite have been found to be still present in equilibrium in theJEV vaccine and can be detected in the following range (n=49):

Free Free Release Results in DS Sulphite Formaldehyde Average (ppm)113.9 41.9 Average (mM) 1.41 1.36 Standard deviation (ppm) 24 14.3 Min(ppm) 66 10.6 Max (ppm) 174 78.7

According to the literature (Ranguelova et al., 2010), transition metalions catalyse the auto-oxidation of (bi)sulphite via sulphur trioxideanion radical (.SO³⁻) formation:

M^(n+)+SO₃ ²⁻→M^((n-1)+)+.SO³⁻

where M may be copper (Cu²⁺), iron (Fe³⁺), oxivanadium (VO²⁺), manganese(Mn²⁺), Nickel (Ni²⁺) or chromate anion (CrO₄ ²⁻) (Alipazaga et al.2004; Berglund et al. 1993; Brandt and Elding 1998; Lima et al. 2002;Shi 1994).

It was shown that such sulphite radicals are highly reactive and canoxidize various substances, such as ascorbate, Hydroquinone andHistidine (Huie at al., 1985). A review of free radical chemistry ofsulphite was published by Neta & Huie, 1985. The authors also show thatradical formation can be also catalysed by photoionization of sulphiteas follows:

SO₃ ²⁻ +hν→.SO₃ ⁻ +e ⁻

Radical formation catalysed by light might also explain differencesobserved in potency and ELISA results of unlabeled naked syringes (usedfor release testing and reference purposes) and fully packaged finalvaccine lot samples for the JEV product. Fully packed samples arecompletely protected from light, whereas unlabeled syringes might beexposed to light during storage and handling.

An important reaction of the sulphite radical in auto-oxidation systemsis with molecular oxygen to form a peroxyl radical which is much morereactive:

.SO³⁻+O₂→.SO⁵⁻

The solubility of O₂ in water at 0° C. and 20° C. is 0.4 mM and 0.25 mM,respectively. Assuming that O₂ solubility in a composition of theinvention is in a similar range, a considerable amount of oxygen ispresent to form the peroxyl radical. This radical is a much strongeroxidant compared to .SO³⁻ and can oxidize certain substrates which arenot attached by .SO³⁻ at all and which, in fact, can form radicals thatoxidize sulphite ions. In such cases, when the redox potential of thesubstrate is intermediate between those of −SO³⁻ and −SO⁵⁻, a reactionchain is likely to develop in presence of O2 following the generalpattern:

SO³⁻+O₂→.SO⁵⁻

SO⁵⁻+X→SO₅ ²⁻+.X⁺

.X⁺+SO₃ ²⁻→X+.SO³⁻

The intermediacy of a substrate X may enhance the chain process ofsulphite oxidation by oxygen. The one-electron reduction of −SO⁵⁻ yieldsHSO⁵⁻ (peroxymonosulfate), a very strong oxidant that is capable tooxidize many organic compounds (Lambeth et al., 1973; Ito & Kawanashi,1991). Peroxymonosulfate is also a precursor sulphate anion radical.SO⁴⁻.

.SO⁵⁻+HSO³⁻→−SO⁴⁻.+HSO⁴⁻

The .SO⁴⁻.radical is a very strong oxidant, nearly as strong as thehydroxyl radical (.OH), and is very likely to oxidize other biomoleculesby one-electron oxidation.

In a preferred embodiment, the composition comprising a proteinaccording to the invention is a therapeutic composition or animmunogenic composition, such as a vaccine. Therapeutic compositions areadministered to individuals, such as a human or an animal. In particularfor such compositions, it is important that the protein within thecomposition still has its therapeutic effect at the time it isadministered to said individual. Degradation of the protein or changesto the protein in its structure may result in the protein to lose itstherapeutic activity. Similarly, degradation or structural changes ofthe immunogenic composition will also lead to a reduction in theeffectivity of the composition in inducing and/or boosting an immuneresponse in an individual. An immunogenic composition is preferablyadministered to an individual to counteract or prevent a viral orbacterial infection. Protein contained within the aqueous immunogeniccomposition can be a single protein or a multimeric protein or part of acomplex comprising said protein (e.g. such as part of a virus or a cell,e.g. bacterial cell). In a preferred embodiment said complex comprises alive attenuated or inactivated virus or bacterium or a immunogenic viralor bacterial protein or an immunogenic part of such protein (e.g. animmunogenic peptide). If said immunogenic composition is administered toprovide protection against a viral or bacterial infection, degradationof protein may result in loss of protective capability of theimmunogenic composition. The term “immunogenic viral or bacterialprotein” refers to a viral or bacterial protein which is capable ofeliciting an immune response. The term “immunogenic part” as used hereinrefers to a part of a viral or bacterial protein which is capable ofeliciting an immune response. Preferably the immune response elicitedrecognizes both said part of the protein and the entire protein.Therefore, in one embodiment, an aqueous composition comprising aprotein according to the invention is an immunogenic composition. Saidcomposition is preferably a therapeutic composition and/or prophylacticcomposition such as a vaccine. Also provided is a vaccine that is anaqueous composition comprising a protein according to the invention

An “immunogenic composition” is herein defined as a composition that iscapable of eliciting an immune response when administered to anindividual. The elicited immune response can be humoral, cellular or acombination thereof and includes, but is not limited to, the productionof antibodies, B cells such as activated B cells, and T cells such asactivated T cells. An immune response as used herein is preferablydirected specifically to one or more immunogens within a compositioncomprising a protein according to the invention. An immunogeniccomposition of the present invention can be administered to anindividual by any technique known in the art including, but not limitedto, intramuscular (IM), intradermal (ID), subcutaneous (SC),intracranial (IC), intraperitoneal (IP), or intravenous (IV) injection,transdermal, oral, intranasal, or rectal administration, andcombinations thereof, preferred are intramuscular (IM), intradermal(ID), subcutaneous (SC), intracranial (IC), intraperitoneal (IP), orintravenous (IV) injection. In a preferred embodiment an immunogeniccomposition comprising a protein according to the invention is used foreliciting an immune response that may be useful in chronic setting (suchas cancer treatment) or prophylactic setting (such as a typicalvaccine). It is preferred that the immunogenic composition is used as avaccine, i.e. prophylactic use. In this embodiment the aluminium istypically present as the adjuvant.

An “adjuvant” as used herein refers to a pharmacological orimmunological agent that modifies the effect of other agents, such as animmunological agent that increases the antigenic response. Adjuvantstypically serve to bring the antigen—the substance that stimulates thespecific protective immune response—into contact with the immune systemand influence the type of immunity produced, as well as the quality ofthe immune response (magnitude or duration); decrease the toxicity ofcertain antigens; and provide solubility to some vaccines components

An “individual” is herein defined as a human or an animal. Individualsinclude but not limited to chickens, ducks, geese, turkeys, swans, emus,guinea fowls and pheasants, humans, pigs, ferrets, seals, rabbits, cats,dogs and horses. In a preferred embodiment of the invention anindividual is a mammal, preferably a human.

An aluminium adjuvant is often prepared by controlled exposure of anaqueous solution of aluminium ions, to alkaline conditions (for reviewsee, Lindblad, EB (2004) Immunol. and Cell Biol. Vol 82: 497-505). Inthe present invention it has been found for the JEV product (seeexperimental part) that a large amount of the heavy metal in thisaqueous solution of aluminium ions ends up in the aluminium saltprecipitate for the aluminium adjuvant. It has further been found thatthe amount of heavy metal in the aluminium precipitate affects thestability of the vaccine during storage of the vaccine. The amount ofheavy metal that is present in the aluminium-salt can thus be controlledby determining the amount of heavy metal in the salt but also, andpreferably, by controlling the amount of heavy metal in the aqueoussolution of aluminium ions. The invention thus further provides a methodfor preparing a clinical grade aluminium-salt precipitate forincorporation into a medicament and/or vaccine, said method comprisingpreparing an aqueous solution of aluminium ion and precipitating saidaluminium-ions from said solution, and determining the level of a heavymetal in the solution and/or the aluminium-salt precipitate, preferablywherein said solution and/or the aluminium-salt precipitate isdetermined to comprise an amount that results in less than 350 ppb heavymetal in the final composition e.g. when re-suspended in the finalcomposition.

Also provided is a pharmaceutical composition comprising a proteinaccording to the invention, optionally further comprising apharmaceutically acceptable carrier and/or diluent. “A pharmaceuticallyacceptable diluent” as used herein is defined as any solution, substanceor combination thereof that has no biologically or otherwise unwantedactivity, meaning that it can be administered to an individual togetherwith other components of an immunological composition without causing asubstantial adverse reaction. Examples of suitable carriers for instancecomprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA orRSA) and ovalbumin. In one preferred embodiment said suitable carriercomprises a solution, like for example saline.

In one aspect, a method according to the invention is used forprolonging the storage life or shelf life of an aqueous compositioncomprising a protein according to the invention. As used herein, theterm “shelf life” is defined as the period of time a compositioncomprising a protein according to the invention can be stored withoutbecoming unsuitable for use, for instance due to degradation of protein(e.g. is within the potency specification of the composition, e.g.vaccine, as required by the regulatory agency that approved or willapprove the vaccine). During storage of aqueous compositions asdescribed herein, degradation of the protein may occur, in particularwhen a certain level (as described herein) of heavy metals is exceeded.Degradation generally increases with time when such aqueous compositionsare stored. Now that it is found that degradation of protein is reducedin an aqueous composition comprising in addition to said protein analuminium-salt, if said composition comprises less than 350 ppb heavymetal based on the weight of the aqueous composition, it has becomepossible to counteract degradation of protein in aqueous compositions.By counteracting degradation of protein with a method according to theinvention, the stability of said protein within said composition isincreased and the storage life of an aqueous compositions comprisingsaid protein is prolonged. An aqueous composition according to thepresent invention provides the advantage that it is stable and does notundergo degradation of protein during a prolonged period. Such aqueouscomposition comprising a protein according to the invention is stablefor at least one month at elevated temperature such as e.g. 20 or 37°C., preferably for at least three months at elevated temperature such ase.g. 20 or 37° C.

An aqueous composition comprising a protein according to the inventionis preferably stored at a temperature of between 0° C. and 20° C. tocontribute to an increased shelf life, more preferably between 2° C. and15° C., more preferably between 2° C. and 10° C., most preferablybetween 2° C. and 8° C. The shelf life at temperature between 2° C. and8° C. is stable preferably for at least three months such as e.g. 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24months, preferably 20 or 24 months, more preferably 20 months at atemperature of between 2 to 8° C. degrees Celsius. Provided is thus anaqueous composition comprising a protein according to the inventionhaving a shelf life of around 12 to 24 months. The invention furtherprovides an aqueous solution according to the invention which has beenstored for at least one month, preferably for at least two months, morepreferably for at least three months, more preferably for at least sixmonths.

As used herein “a stable protein composition” means that when comparedto a starting composition not more than 50%, preferably not more than40%, even more preferably not more than 30%, even more preferably notmore than 20%, even more preferably not more than 10, even morepreferably not more than 5% of the protein in said composition isdegraded. “Degraded” in this context refers to any detectablemodification of the protein when compared to the protein in the startingcomposition. For instance, a decrease in detection of the protein with amonoclonal antibody such as e.g. an antibody recognizing a neutralizingepitope is suitable and can be measured with any method known in theart, such as ELISA (see Example 4). The level detected can for instancebe compared with the level detected with a polyclonal specific for thesame protein such as e.g. representing the amount of total protein(changed or unchanged). Also provided is a method of prolonging theshelf life of an aqueous composition comprising a protein and analuminium-salt, said method comprising selecting and/or preparing analuminium-salt resulting in an aqueous composition with a heavy metalcontent of less than 350 ppb based on the basis of weight of the aqueouscomposition and combining said aluminium salt, said protein and water toproduce said aqueous composition.

Further provided is a method of improving the shelf life reproducibilityof preparations of aqueous compositions comprising a protein and analuminium-salt, said method comprising obtaining at least two differentaluminium-salt preparations, determining the amount of at least oneheavy metal in said aluminium-salt preparations, selecting from saidaluminium-salt preparations, aluminium-salt preparations that compriseless than 350 ppb of said at least one heavy metal; and combiningaluminium salt of said selected preparations with said protein and waterto produce said aqueous compositions.

In another aspect the invention provides a method for analysing thestorage stability of a composition comprising aluminium and atherapeutic or prophylactic compound, said method comprising combininginto a composition a pre-determined amount of therapeutic or vaccine anda pre-determined amount of an aluminium salt, said method furthercomprising storing said composition for at least 2 weeks, preferably atleast 4 weeks and preferably at least one month, at a temperature ofmore than 20° C., preferably at a temperature of about 22° C. anddetermining the stability and/or the amount of protein, preferablytherapeutic or prophylactic compound, in said composition. Asdemonstrated in Examples 1 and 2, a temperature of 22° C., which ishigher temperature compared to normal storage conditions or about 2-8°C., results in accelerated degradation of protein in an aqueouscomposition comprising protein, an aluminium-salt and more than 350 ppbof said heavy metal based on weight with respect to said composition.Thus, a temperature of about 22° C. and a storage duration of at least 2weeks, preferably at least 4 weeks, are suitable to determine thestorage stability of aqueous compositions comprising a protein accordingto the invention. The storage stability can be determined by any methodknown in the art. The storage stability of an aqueous compositioncomprising a protein according to the invention is preferably analyzedby determining the storage stability of said protein, preferably bydetermining a storage sensitive epitope on said protein. For instance,as described in Example 1 and 2, the stability of a protein, preferablyan antigen, is determined by determining the ratio of intact storagesensitive epitope, such as intact antigenic epitope (e.g. epitope of aneutralizing epitope) content, and total protein content. “Intactstorage sensitive epitope” or “intact antigenic epitope” as used hereinmeans that degradation has not occurred within said epitope. The intactantigenic epitope content is for instance measured by determiningprotein bound to a monoclonal antibody specifically directed againstsaid epitope in, for example, an ELISA. The total protein content is forinstance measured by determining protein bound to polyclonal antibodywhich is directed against various epitopes within the protein, forexample by ELISA. The relative specific epitope content can then beexpressed as the ratio of the total antigen content determined bybinding to monoclonal antibody divided by total antigen contentdetermined by binding to polyclonal antibody. A high ratio indicateshigh antigenic epitope content and a low ratio indicates a low antigenicepitope content. A low ratio measured for an aqueous composition afterstorage at least 20° C., preferably 22° C., for at least 2 weeks,preferably 4 weeks, as compared to the ratio measured for said aqueouscomposition before storage, indicates that structural changes have takenplace within the antigenic epitope. Structural changes within saidantigenic epitope indicate reduced storage stability of the aqueouscomposition.

The heavy metal content of an aqueous composition prepared according tothe invention is less than 350 ppb based on the weight of the aqueouscomposition. Generally, the less heavy metal such aqueous compositioncontains, the less degradation of protein occurs. Preferably, therefore,the heavy metal content is less than 325 ppb based on the weight of theaqueous composition, more preferably less than 300 ppb, more preferablyless than 275, more preferably less than 250 ppb and more preferablyless than 235 ppb based on the weight of the aqueous composition.

As used herein the term “heavy metal” refers to the total amount ofelements that exhibit metallic properties and includes the transitionmetals, metalloids, lanthanides, and actinides. Transition metals areelements whose atom has an incomplete d sub-shell, or which can giverise to cations with an incomplete d sub-shell, and include zinc,molybdenum, cadmium, scandium, titanium, technetium, palladium,vanadium, chromium, manganese, iron, cobalt, rhodium, hafnium, copper,nickel, ytrrium, niobium, ziorconium, rughenium, silver, tantalum,rhenium, thungsten, osmium, meitnerium, platinum, iridium, mercury,bohrium, seaborgium, hassium. Metalloids are Boron (B), Silicon (Si),Germanium (Ge), Arsenic (As), Antimony (Sb), Tellurium (Te), Polonium(Po). The lanthanides are the fifteen metallic chemical elements withatomic numbers 57 through 71, i.e. Lanthanum, Cerium, Praseodymium,Neodymium, Promethium, Samarium, Europium, Gadolinium, Terbium,Dysprosium, Holmium, Erbium, Thulium, Ytterbium and Lutetium. Theactinides are the fifteen metallic chemical elements with atomic numbersfrom 89 to 103, actinium, thorium, protactinium, uranium, neptunium,plutonium, americium, curium, berkelium, californium, einsteinium,fermium, mendelevium, nobelium and lawrencium. Preferably said heavymetal is selected from the transitional metals. In another preferredembodiment, said heavy metal is a metal having a molar mass of between21 and 83, more preferably from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V,Cr, Pb, Rb and Mo. In an even more preferred aspect, the heavy metal isselected from the heavy metals Cu, Ni, and Fe.

As demonstrated in the Examples, Aluminium hydroxide (Alum) Lot 4230 wasidentified to contribute significantly to antigen degradation in JEVvaccine FVL09L37. In Example 3 it is shown that this Alum lot comprisesat least the following metals: Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V.Higher levels of Fe, Ni and Cu ions were noted in Alum lot 4230 whencompared to other investigated lots. Lot 4230 was the only one whereresidual Cu ions were detected. Therefore, preferably said heavy metalis selected from the group consisting of Cu, Ni, W, Co, Os, Ru, Cd, Ag,Fe, V, more preferably from Fe, Ni and Cu.

The amount of heavy metal in an aqueous composition of the invention isdefined herein above. This amount is typically for the total ofdetermined heavy metals, or for the heavy metals Fe, Cr and Ni, or acombination thereof which constitute the major heavy metals by weight inthe aqueous composition of the invention. For specific heavy metalsdifferent maximums amounts may be preferred. For instance, it ispreferred that the amount of Fe in the aqueous composition of theinvention is less than 350 ppb based on the weight of the aqueouscomposition. In a preferred embodiment the amount of Fe is less than 250ppb Fe based on the weight of the aqueous composition.

There is strong evidence that many of the pro-inflammatory effects ofaluminium adjuvants are mediated via the formation of reactive oxygenspecies (ROS). Aluminium can, under physiological conditions, promotethe reduction of Fe(III) to Fe(II) and the oxidation of the latter. Thusthe combination of Fe and Al in the adjuvant will potentiate theformation and activities of ROS (Exley, C (2010). Trends in Immunol.Vol. 31: pp 103-109). In the present invention it has been found that Fecan be present in an aqueous composition of the invention withoutsignificantly affecting the storage stability of the composition. Inthis embodiment of the invention it is preferred that the aqueouscomposition of the invention comprises between 5 ppb and 250 ppb Febased on the weight of the aqueous composition. In these amounts theformation of ROS during storage due to the presence of such amount of Fe(if any ROS) does not significantly affect the storage stability of theaqueous composition as defined elsewhere herein. However, the amountsare sufficient to allow pro-inflammatory effects followingadministration of the vaccine in vivo.

In the present invention it has been found that particularly thepresence of heavy metal Cu seriously affects the storage stability ofthe composition of the invention. In a particularly preferred embodimentan aqueous composition of the invention therefore comprises less than 3ppb Cu based on the weight of the aqueous composition. Preferably lessthan 2.5 ppb. In a particularly preferred embodiment said aqueouscomposition comprises Cu at a level that is below the detection limit ofthe method for the detection of copper as described in the Examples.

In the present invention it has been found that particularly the heavymetal Ni affects the storage stability of the composition of theinvention. In a particularly preferred embodiment an aqueous compositionof the invention therefore comprises less than 40 ppb Ni based on theweight of the aqueous composition. Preferably less than 30 ppb, morepreferably less than 20 pbb and more preferably less than 15 pbb Nibased on the weight of the aqueous composition. In a particularlypreferred embodiment said aqueous composition comprises Ni at a levelthat is below the detection limit of the method for the detection ofnickel as described in the Examples.

The heavy metal can be present in electronic neutral form or it can beionised. Typically and preferably the heavy metal is present in ionicform in an aqueous composition of the invention.

Metal content of a composition can be determined in various ways. In oneaspect, a method according to the invention comprises determining thelevel of a heavy metal in an aqueous composition and/or thealuminium-salt present in said aqueous composition. Methods formeasuring the level of one or more heavy metals in an aqueous solutionare known in the art. Examples of such methodsinductively-coupled-plasma mass spectrometry (ICP-MS), flame atomicabsorption spectrometry (F-AAS), and/or graphite furnace atomicabsorption spectrometry (GF-AAS).

An example of an assay which can be used to determine the content ofheavy metals is described in Example 3. The assay involves treating asample of an aqueous solution containing an Aluminum-salt withconcentrated HNO₃ under heat until a clear solution is obtained. Theclear solution can then be further diluted and analyzed, for instance byICP-MS, F-AAS and/or GF-AAS., for the presence and content of metal ionsincluding Pb, Cd, Cr, Co, Fe, Cu, Ni, Ag, W and Al.

Examples 1 and 2 demonstrate that the JEV antigen shows higher stabilityat pH 7.5-8 as compared to pH 7. In the Examples antigen stability isexpressed as the ratio of monoclonal/polyclonal ELISA. The monoclonalantibody used (clone 52-2-5) was shown to recognize a neutralizingepitope in the Japanese Encephalitis Vaccine (JEV). The relativespecific epitope content can be expressed as the ratio of the totalantigen content determined by “monoclonal ELISA” divided by totalantigen content determined by “polyclonal ELISA”. Without being bound totheory, the effect of a higher antigen stability at pH 7.5-8 can beexplained based on the underlying assumed complex reaction chemistry ofsulphites. pH might influence the related to equilibrium reactionconditions of the sulphite/formaldehyde reaction and surface charge ofcertain proteins/amino acid side chains accessible to modification. ThepH may affect oxidation by direct influence on redox potentials of theamino acid residues and the oxidizing agents, e.g. free radicals.Therefore, in one embodiment, a method according to the inventioncomprises buffering said aqueous composition at a pH of between 7.5 and8.5.

Various aluminium salts are being used in compositions foradministration of an individual. Aluminium adjuvant typically containsan aluminium oxide or sulphate or a combination thereof. In a preferredembodiment the aluminium salt comprises aluminiumoxide (Al₂O₃),aluminiumhydroxide (Al(OH)₃) or aluminiumphosphate (AlPO₄).

In a preferred embodiment the aqueous composition of the inventionfurther comprises a reactive compound. Typically though not necessarilythe reactive compound is present as a result of a manipulation of theaqueous composition, for instance to treat or inactivate infectiousagent if any in the composition. The reactive compound can also bepresent for another reason. Sulphite, for instance, is sometimes presentto inactivate any residual formaldehyde in the aqueous solution.Formaldehyde is typically a chemical that is often used to inactivateany infectious agent.

The reactive compound is preferably a redox active compound, radicalbuilding compound and/or a stabilizing compound. In a preferredembodiment said aqueous composition of the invention comprisesformaldehyde, ethanol, chloroform, trichloroethylene, acetone, TRITON™X-100 (Polyethylene glycol tert-octylphenyl ether), deoxycholate,diethylpyrocarbonate, sulphite, Na₂S₂O₅, beta-proprio-lacton,polysorbate such as TWEEN® 20 (Polysorbate 20) or, TWEEN® 80(Polysorbate 80), O₂, phenol, PLURONIC (poloxamer) type copolymers, or acombination thereof.

Sulphite is preferably present in an amount of between 0.1 mM and 5 mM,or preferably between 0.5-2 mM. Formalin is preferably present in anamount of between 0.1 mM and 5 mM, more preferably between 0.5 mM and 2mM. Oxygen is preferably present in an amount that is equivalent to thesolubility of O₂ at the measured temperature, O₂ is preferably presentin an amount of between 10 and 250 uM when measured at 20 degreesCelsius. When measured at a temperature of 0 degrees Celsius O₂ ispreferably present in an amount of between 10 and 400 uM. A stabilizingcompound is preferably present in an amount of between 10 and 400 uM.Similarly a redox active compound is present in an amount of between 0.1mM and 5 mM, or preferably between 0.5-2 mM. A radical building compoundis preferably present in an amount of between 0.1 mM and 5 mM, orpreferably between 0.5-2 mM. In this context and for the sake of clarityit is important to note that redox active compound, the radical buildingcompound and/or the stabilizing compound is consumed in the productionof a radical, whereas the heavy metal is indicated herein above, is acatalyst in the production of a radical and is not consumed, as such.The redox active compound, the radical building compound and/or thestabilizing compound is therefore not a heavy metal.

The total amount of redox active compound, the radical building compoundand/or the stabilizing compound although small in absolute amounts canstill be significant in relation to the antigen or protein in theaqueous composition of the invention. The antigen/protein is preferablypresent in an amount of between 0.1 nmol to 1 umol, more preferablybetween 1 nmol and 100 nmol.

The concentration of protein, preferably a therapeutic or vaccineprotein, in an aqueous composition comprising a protein according to theinvention is preferably between 1 ng/ml and 10 mg/ml, preferably between10 ng/ml and 1 mg/ml, more preferably between 100 ng/ml and 100 ug/ml,such as between 1 ug/ml and 100 ug/ml. The concentration is preferablyat least 1 ng/ml to ensure that the therapeutic or vaccine protein is ina concentration sufficient to exert its therapeutic effect whenadministered to an individual. The concentration should, however,preferably not exceed 10 mg/ml in order to prevent or reduce theoccurrence of possible side effects associated with administration ofsaid protein to an individual. In particular, the concentration of viralprotein in an aqueous composition according to the invention comprisingJEV is preferably between 0.01 μg/ml and 1 mg/ml, more preferablybetween 0.1 μg/ml and 100 ug/ml. In an exemplary embodiment, of theinvention, an aqueous composition according to the invention comprisesabout 10 ug/ml of JEV. The dose of a single administration of an aqueouscomposition comprising a protein, preferably a therapeutic or vaccineprotein, according to the invention is preferably between 0.1 ml and 10ml, preferably between 0.5 ml and 5 ml, such as 0.5 ml, 1 ml, 1.5 ml, 2ml, 2.5 ml, because such dose allows for convenient administration to anindividual, such as a human.

A method according to the invention is preferably used to increasestability of an immunogenic composition, preferably a vaccine,comprising an aluminium-salt based adjuvant. Non-limiting examples ofsuch vaccines are those directed against infection with Bacillusanthracis (causing Anthrax), Corynebacterium diphtheriae (causingdiphteria), Clostridium tetani (causing tetanus), Pseudomonas such asPseudomonas aeruginosa, Staphylococcus such as Staphylococcus aureus orStaphylococcus epidermidis, Haemophilus influenzae type B bacteria(Hib), polio virus, hepatitis A virus, hepatitis B virus, HumanPapillomavirus, influenza virus, Japanese encephalitis virus, Rotavirus,Rickettsiae bacteria (causing Typhus), yellow fever virus, VaricellaZoster Virus, Meningococcus, or combinations thereof, such as, but notlimited to, DTP (diphteria, tetanus, polio). An aqueous compositioncomprising a protein according to the invention therefore preferablycomprises a protein which is a viral or bacterial protein, preferably aprotein of Bacillus anthracis, Corynebacterium diphtheriae, Clostridiumtetani, Haemophilus influenzae type B bacteria (Hib), polio virus,hepatitis A virus, hepatitis B virus, Human Papillomavirus, influenzavirus, Japanese encephalitis virus, Rotavirus, Rickettsiae bacteria,yellow fever virus, Varicella Zoster Virus and/or Meningococcus. In apreferred embodiment, said protein contained in a composition comprisinga protein according to the invention is a viral protein from a virus ofthe Flaviviridae family, preferably of a Japanese encephalitis virus(JEV). As demonstrated in the Examples, the stability of aqueouscompositions comprising a JEV protein, an aluminium-salt and comprisingless than 350 ppb heavy metal based on the weight of the aqueouscomposition, and in particular wherein the amount of Cu is less than 3ppb based on the weight of the aqueous composition, is increased ascompared to aqueous composition comprising more than 350 ppb of heavymetal and more than 3 ppb of Cu. Thus, a method according to theinvention is particularly suitable to increase stability of an aqueouscomposition comprising a JEV protein.

In another preferred embodiment or aspect of the invention, said proteincontained in a composition comprising a protein according to theinvention is a bacterial protein from a bacterium of the Pseudomonasfamily, preferably of Pseudomonas aeruginosa. As demonstrated in theExamples, the stability of aqueous compositions comprising Pseudomonasaeruginosa fusion protein (SEQ ID NO: 1) and an aluminium-salt isreduced when more than 350 ppb heavy metal based on the weight of theaqueous composition is present.

An aqueous composition comprising a protein according to the inventionis particularly suitable for use as an immunogenic composition orvaccine. For instance, such compositions are particularly useful forimmunize an individual to treat or prevent a viral or bacterialinfection. In one embodiment, the invention therefore provides a methodfor the treatment of an individual comprising obtaining an immunogenicaqueous composition comprising a protein and an aluminium-salt, saidaluminium-salt having less than 350 ppb heavy metal based on the weightof the aqueous composition, preferably less than 3 ppb of Cu, andadministering the immunogenic aqueous composition to an individual inneed thereof. Also provided is a method for the prophylactic treatmentof an individual comprising obtaining an immunogenic aqueous compositioncomprising a protein and an aluminium-salt, said aluminium-salt havingless than 350 ppb heavy metal based on the weight of the aqueouscomposition, preferably less than 3 ppb of Cu, and administering theimmunogenic aqueous composition to an individual in need thereof.Further provided is a method for inducing and/or boosting an immuneresponse towards an antigen in an individual, said method comprisingobtaining an aqueous composition comprising a protein comprising saidantigen and an aluminium-salt, said aluminium-salt having less than 350ppb heavy metal based on the weight of the aqueous composition,preferably less than 3 ppb of Cu, and administering the aqueouscomposition to an individual in need thereof. In another aspect theinvention provides a method for immunizing an individual comprisingadministering to said individual at least two immunogenic compositionsat an interval of at least two weeks between each administration, andwherein each of said at least two immunogenic compositions comprise thesame antigen, and wherein at least one of said immunogenic compositionsfurther comprises an aluminium salt having less 350 ppb heavy metalbased on the weight of the aqueous composition, preferably less than 3ppb of Cu, and administering the immunogenic aqueous composition to anindividual in need thereof. Nucleic acid compositions are sometimes alsoadministered together with aluminium. Thus for the present invention itis possible to replace “protein” in an aqueous composition of theinvention with nucleic acid. Thus in one embodiment the inventionprovides a method for preparing an aqueous composition comprisingaluminium and a nucleic said method comprising—combining analuminium-salt, said nucleic acid and water to produce said aqueouscomposition and—determining the level of a heavy metal in the aqueouscomposition and/or the aluminium-salt. The invention also provides amethod for preparing an aqueous composition comprising aluminium and anucleic acid said method comprising—preparing or selecting analuminium-salt having less 350 ppb heavy metal based on the weight ofthe final aqueous composition, preferably less than 3 ppb of Cu, andadministering the immunogenic aqueous composition to an individual inneed thereof and

-   -   combining said aluminium salt, said nucleic acid and water to        produce said aqueous composition. In a preferred embodiment said        methods further comprising buffering said aqueous composition at        a pH of between 7.5 and 8.5. In a particularly preferred        embodiment said methods, further comprise packaging aliquots of        said aqueous composition having less than 350 ppb heavy metal        based on the weight of the aqueous composition in separate        air-tight storage containers. The nucleic acid may be        administered for therapeutic purposes. For instance, in the form        of an antisense RNA, RNAi or mimic thereof. The nucleic acid may        also be administered in the form of an infectious agent,        typically a virus or a modified virus, as is the case in many        gene therapy approaches. In that case the nucleic acid is        enclosed in a particle that comprises protein. The invention        thus further provides an aqueous composition comprising a        nucleic acid and an aluminium-salt, said composition comprising        less than 350 ppb heavy metal based on the weight of the aqueous        composition.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

REFERENCES

-   Alipazaga M V, Moreno R G M, Coichev N. 2004. Synergistic effect of    Ni(II) and Co(II) ions on the sulphite induced autoxidation of    Cu(II)/tetraglycine complex. Dalton Trans 13:2036-2040.-   Arunee Wittayanukulluk, Dongping Jiang, Fred E. Regnier, Stanley L.    Hem, “Effect of microenvironment pH of aluminum hydroxide adjuvant    on the chemical stability of adsorbed antigen”, Vaccine 22 (2004)    1172-1176-   Berglund J, Fronaeus S, Elding L I. 1993. Kinetics and mechanism for    manganese-catalyzed oxidation of sulfur(IV) by oxygen in aqueous    solution. Inorg Chem 32:4527-4538.-   Brandt C, Elding L I. 1998. Role of chromium and vanadium in the    atmospheric oxidation of sulfur (IV). Atmos Environ 32(4):797-800.-   Exley, C (2010). Trends in Immunol. Vol. 31: pp 103-109.-   Ito, Kimiko and Kawanashi, Shosuke. Site-specific fragmentation and    modification of Albumin by sulphite in presence of metal ions or    peroxidase/H₂O₂: Role of Sulphate radical. Biochem and Biophys Res    Comm., 1991, 176, 1306-1312-   Huie R. E., Neta P. One-electron redox reaction in aqueous solutions    of sulphite with hydroquinone and other hydroxyphenols. J. Phys.    Chem., 1985, 89 (18), 3918-3921-   Kalina Ranguelova, Marcelo G. Bonini, and Ronald P. Mason:    (Bi)sulphite Oxidation by Copper, Zinc-Superoxide Dismutase:    Sulphite-Derived, Radical-Initiated Protein Radical Formation.    Environmental Health Perspectives 2010, 118 (7), 970-975-   Lampeth D. O., Palmer G. The kinetics and mechanism of reduction of    electron transfer proteins and other compounds of biological    interest by dithionite. J. Biochem. Chem. 1973, 248, 6095-6103-   Li S, Schöneich C, Borchardt R T. Chemical instability of protein    pharmaceuticals: Mechanisms of oxidation and strategies for    stabilization. Biotechnol Bioeng. 1995 Dec. 5; 48(5):490-500-   Lindblad, E B (2004) Immunol. and Cell Biol. Vol 82: 497-505.-   Lima S, Bonifacio R L, Azzellini G C, Coichev N. 2002. Ruthenium(II)    tris(bipyridyl) ion as a luminescent probe for oxygen uptake on the    catalyzed oxidation of HSO³⁻. Talanta 56:547-556.-   Mayo J C, Tan D X, Sainz R M, Natarajan M, Lopez-Burillo S, Reiter    R J. Protection against oxidative protein damage induced by    metal-catalyzed reaction or alkylperoxyl radicals: comparative    effects of melatonin and other antioxidants. Biochim Biophys Acta.    2003 Mar. 17; 1620(1-3):139-50.-   Neta P., Huie R. E.: Free Radical Chemistry of Sulphite.    Environmental Health Perspectives 1985, 64, 209-217-   Shi X. 1994. Generation of −SO³⁻ and OH radicals in SO₃ ²⁻ reactions    with inorganic environmental pollutants and its implications to SO₃    ²⁻ toxicity. J Inorg Biochem 56(3):155-165.-   Stadtman E R. Metal ion-catalyzed oxidation of proteins: biochemical    mechanism and biological consequences. Free Radic Biol Med. 1990;    9(4):315-25.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RP-HPLC elution profiles of chlorobutyl stopper extract (1:4diluted) and JEV09L37 SN.

FIG. 2: SEC HPLC elution profiles of PS (2 mg/mL) before and aftertrypsin cleavage.

FIG. 3: SEC HPLC elution profiles of Trypsin treated PS and degraded PSas present in NIV11A74. Note that elution profiles were normalized tosimilar peak height to allow better comparison.

FIG. 4: DOE evaluation of the ratio monoclonal/polyclonal ELISA byPareto chart analysis and main effects plots (4 weeks at 22° C.).

FIG. 5: DOE evaluation of the ratio monoclonal/polyclonal ELISA byPareto chart analysis and main effects plots (8 weeks at 22° C.).

FIG. 6: Contour plot of the estimated response

FIG. 7: Residual plot of the estimated response

FIG. 8: ELISA ratio (monoclonal/polyclonal) for JEV formulations at pH 7in presence of Ni, Cu and Cr. Samples were stored at 22° C. for 5 weeks

FIG. 9: Summary of results obtained after 7 weeks at 22° C. Shown arethe raw data of ratio as a function of pH and metal ion type andcombined results for each parameter.

FIG. 10: Mean ratio of DP formulations prepared with different Alumlots. Samples were stored for 6 weeks at 22° C. Error bars represent the95% confidence interval calculated based on pooled standard deviation.Samples from left to right: Alum 3877, Alum 4074, Alum 4230 nonGI, Alum4230 GI, Alum 4470, Alum 4563, Alum 4621, Alum Mix 4074_4230.

FIG. 11: Particle size distribution of ALHYDROGEL® samples.

FIG. 12: ALHYDROGEL® titration curves in PBS. ▪ non-irradiated AlOH(RQCS0890), ▴ GI AlOH (RQCS1200), ♦ GI AlOH (RQCS1342), + GI AlOH(RQCS0448)

FIG. 13: Overview of tested ALHYDROGEL® batches. Total concentration ofcontaminating metal ions in ng/mL and share of the main metal ions Fe,Cr and Ni are shown.

FIG. 14: Amino acid sequence of Ala-(His)6-OprF190-342-OprI21-83 (SEQ IDNO: 1)—herein also referred as “protein A”.

EXAMPLES Example 1

Aluminium hydroxide (Alum) Lot 4230 was previously identified tocontribute significantly to antigen degradation in FVL09L37. In thisparticular Alum lot much higher residual metal ion content was observedcompared to other Alum lots used for formulation of the inactivated JEVantigen. This Example demonstrates additional studies carried out tofurther identify the underlying root-cause mechanism and influence ofmetal ions on degradation pathway of JEV. Design of experiments (DOE)was performed to work out the influence of individual parameters onantigen stability.

Parameters tested in a 25 full factorial DOE were

-   -   Aluminium hydroxide Lot 4230 vs. Aluminium hydroxide Lot 4074    -   Presence of excess Protamine sulfate fragments    -   Presence of leachables from chlorobutyl rubber stopper    -   pH range 7 to 8    -   Residual formaldehyde content

Alum lot 4230 contains much higher levels of residual metal ionimpurities compared to other Alum lots used for formulation of JEV. A“Design-of-Experiment” (DOE) was selected to further investigate thepotential root cause mechanism and interaction of parameters thatfinally could lead to product degradation. In factorial designs,multiple factors are investigated simultaneously during the test. As inone factor designs, qualitative and/or quantitative factors can beconsidered. The objective of these designs is to identify the factorsthat have a significant effect on the response, as well as investigatethe effect of interactions (depending on the experiment design used).Predictions can also be performed when quantitative factors are present,but care must be taken since certain designs are very limited in thechoice of the predictive model. For information about DOE in general see(Siebertz, Karl; van Bebber, David, Hochkirchen, Thomas: StatistischeVersuchsplanung: Design of Experiments (DoE). Publisher: Springer BerlinHeidelberg; 1st Edition (2010), ISBN-10: 3642054927).

1.1 DOE Study Design 1.1.1 Definition of Parameters and Levels for DOEDesign

The following parameters and levels were taken into consideration fordesigning an appropriate DOE experiment:

-   -   Residual metal ion content of Alum: Aluminium hydroxide Lot 4230        and Lot 4074 were selected as representative of the two extremes        quality with regard to residual metal ions content of Aluminium        hydroxide. The center point was a mixture of 50/50% of both Alum        lots. Initial analysis for remaining metal ion impurities in 2%        Aluminium hydroxide stock solution by ICP-MS showed significant        differences in Cr, Fe, Ni and Cu ion content between these two        lots (see Table 1).    -   Protamine Sulphate fragments: Protamine sulfate (PS) fragments        are present at low quantity (<5 μg/mL) in the final vaccine lot.        It was tested if PS fragments could contribute to virus surface        modification (e.g. interaction/covalent linkage to the virus        surface proteins) in combination with Alum and other factors        used in this study. Therefore a stock solution of PS fragments        was prepared by digestion with Trypsin followed by heat        inactivation and ultrafiltration using a 5 kDa membrane for        protease inactivation and removal of the enzyme. This stock        solution was used for spiking additional PS fragments into the        respective formulations at the high level of 50 μg/mL. In low        level samples no additional PS fragments were spiked and the        actual level in formulations was <5 μg/mL according to HPLC        analysis.    -   pH: Lower and upper level of pH in formulations was 7 and 8 with        the center point at pH 7.5.    -   Leachables/Extractables from syringe plunger: Syringe plungers        (made of chlorobutyl PH701/50 black) that are currently used in        the container closure system. It was tested if leachables from        the chlorobutyl rubber in the formulation could contribute to        antigen modification. Therefore a stock solution of leachables        was prepared and used for spiking experiments. The high level of        spiked leachables in formulation was estimated to be on average        1.4× higher compared to commercial Final Vaccine Lot (FVL). Due        to the harsh extraction conditions additional peaks were        detected not present in FVL samples. Therefore the spiked        formulations represent a “worst case” with regard to leachables        and extractables. Formulations at the low level did not contain        any leachables from chlorobutyl rubber.    -   Residual formaldehyde: For low level formulations no additional        formaldehyde was spiked into the formulation samples. The lower        level was the residual formaldehyde that was still present in        diluted NIV sample after inactivation/neutralization and 2-fold        dilution was in the range of approx. 37 ppm (recalculated from        commercial DS release GMP analytical certificate). For the high        level additional 40 ppm formaldehyde were spiked into the        corresponding formulation (total final content approx. 77 ppm).        It was tested if residual formaldehyde in combination with        higher level of metal ions present in Alum 4230 and possible        other factors could further react with the virus leading to        hyper-cross linking of surface proteins and loss of relevant        epitopes.        Determination of Other Residual Process Related Impurities:        Residual formaldehyde, sulphite and sucrose in final        formulations were estimated based on GMP certificates for        commercial drug substance JEV11A74. Results were recalculated by        the actual 2-fold dilution of NIV to DS used within the DOE        experiments.

Residual Sulphite:

The concentration of residual sulphite was constant in all formulationswith approx. 93 ppm.

Residual Sucrose:

The concentration of residual sucrose was constant in all formulationswith approx. 1% v/w.

1.1.2 DOE Design

These 5 factors were combined in a 25 DOE plan resulting in a totalnumber of 34 experiments including 2 center points with the followingbase design. DOE planning and evaluation were carried out with anappropriate software (Statgraphic Plus 3.0)

Base Design: Factorial 25

Number of experimental factors: 5Number of blocks: 1Number of responses: 1Number of centerpoints per block: 2Number of runs: 34Error degrees of freedom: 18

Randomized: Yes

Factors Low High Units Continuous¹⁾ pH 7.0 8.0 Yes Alum²⁾ 0.0 100.0 Alum4230% Yes Spiked PS Fragments³ 0 50 μg/mL No Spiked leachables ⁴⁾ 0 1.4relative content No compared to FVL Spiked Formaldehyde⁵⁾ 0 40 ppm NoResponses Units ELISA (monoclonal, polyclonal) of desorbed antigen AU/mL¹⁾Continuous means that a center point (mean value from high and lowlevel) is present in the study design. No Center point means that onlylow and high levels are present in the study design. ²⁾Low level (0%)means that formulation was prepared with Alum Lot 4074. High level(100%) means that formulation was prepared with Alum lot 4230. Forcenter point formulations an equal mixture (50/50%) of both Alum lotswas used. ³Since PS is present in NIV used for preparation of drugproduct samples, actual PS concentration in non-spiked formulations was<5 μg/mL and ~50-55 μg/mL for PS-spiked formulations. ⁴⁾ No leachableswere assumed in non-spiked formulations since samples wereprepared/stored in low-bind Eppendorf tubes. Total content of leachablesfrom chlorobutyl rubber syringe plunger in spiked samples was approx.1.4 times higher compared to FVL. ⁵⁾Actual formaldehyde concentration innon-spiked DP samples was approx. 37 ppm, total formaldehydeconcentration in spiked samples was approx. 77 ppm.

2 DEFINITIONS & ABBREVIATIONS AcCN Acetonitrile

DOE Design of experimentsDS Drug substance

FVL Final Vaccine lot

HPLC High performance liquid chromatographyICP-MS Inductively-coupled-plasma mass spectrometryPS Protamine sulphate

RP Reversed Phase

SEC Size exclusion chromatography

SN Supernatant

TFA Trifluoroacetic acidw/o without

3 Materials and Methods 3.1 DOE Studies 3.1.1 Materials

-   -   Syringe plunger stoppers: PH701/50/C black Sil6 7002-1051        (obtained from West, Order No. 2116)    -   100 mL Glass bottle (Schott)+Teflon coated screw cap    -   Aluminium foil    -   HQ water    -   Electrical water bath (IKA, HBR 4 digital)    -   LoBind Eppis 2 ml (Eppendorf, Cat. No. 0030 108.132)    -   Speed Vac (Christ, RVC-2-25)    -   HPLC vials, clear glass, 900 μL, Chromacol (VWR, Cat. no.:        548-1124)    -   HPLC vials, PP, 900 μL, (Agilent, Item no. 5182-0567)    -   Caps for HPLC vials, pre-cut (VWR, Cat. no.: 548-1260)    -   15 ml Falcon tubes (Greiner, Cat. No. 188724)    -   Alum batch 4470 (RQCS 1342); Alum Lot 4230 (RQCS 1200)    -   10×PBS (Gibco, Order No. 14200-091)    -   Parafilm    -   Waters Atlantis T3 column; 3 μm particle diameter; column        diameter/length 2.1×100 mm (Order No. 186003718; Lot 0107372331)    -   Acetonitril (Merck, Cat No. 1.13358.2500)    -   TFA (Sigma, Order No. 302031    -   HPLC system Dionex 3000    -   Solvent Rack SR-3000    -   Pump UltiMate-3000, analytical low pressure gradient pump    -   Autosampler WPS-3000 TSL, analytical autosampler—temperature        controlled    -   Column compartment TCC-3200, temperature controlled    -   PDA-Detector PDA-3000    -   Formaldehyde solution 37% (Merck, Cat No. 1.040031000)    -   Protamine sulphate (Intercell Biomedical Ltd, Batch no. 086056)    -   Ultrafilatrion device (Amicon® Ultra 3 kDa) (Millipore, Cat No.        UFC900324)    -   Incubator Infors HT Incubator Multitron Standard (InforsAG)    -   Trypsin (Sigma, Order No: T0303)        3.1.2 Procedure for Preparation of Extractables from Syringe        Plunger

Syringe plunger (made of chlorobutyl PH701/50 black) that are currentlyused in the FVL container closure system were obtained from West(Germany). Therefore a stock solution of leachables was prepared by heattreatment of syringe plunger in water (90° C./2 h) followed byconcentration in a speed-vac. The relative content of leachables in thisstock solution was estimated by RP-HPLC using a C18 column (Atlantis T3column) and compared to FVL JEVO9L37 supernatant.

Extraction Method

A 100 mL Schott glass bottle with a Teflon coated screw top and a pieceof aluminum foil were washed with hot water and thoroughly rinsed withHQ water. 30 stoppers were filled in the bottle and 30 ml of HQ-waterwere added. The bottle was closed with the aluminum foil fitted betweenbottle and screw and sealed additionally with Parafilm. The bottle washeated in the water bath to 90° C. for 2 hours and allowed to cool toroom temperature. The extract was transferred to 14 low-bind Eppendorftubes (a total of 28 mL extract was recovered). Twelve vials (total of24 mL) were concentrated in a Speed Vac for approximately 44 hours andpooled into a falcon tube to obtain 6 ml of 4× concentrated stopperextract. A control sample containing 30 mL HQ water w/o stopper wasprepared in the same way to evaluate any possible contamination.

C18 RP-HPLC Method

Leachables were Separated by RP-HPLC C18 Column (Atlantis T3) Operatedat 40° C. and 0.25 mL/min. Solvent A was 0.1% TFA in H2O, solvent B was0.1% TFA in AcCN. Separation was performed by linear gradient rangingfrom 0 to 95% B in 30 min. Detection was done at 214 nm, 254 nm and 280nm. The total relative concentration of concentrated stopper extract wasestimated to be 80 fold higher compared to peaks detected in FinalVaccine Lot supernatant (FVL SN; obtained by removal of Alum particlesby centrifugation at 5000 g/5 min) as detected at 254 nm. Therefore atotal relative content of 80 U/mL (arbitrary Units U) were assigned forthe stock solution, whereas the total relative concentration ofleachables in FVL SN was set to 1 U/mL. For DOE studies, the stocksolution was diluted 16-fold into the respective formulations yieldingapprox. 5 U/mL of total extractables.

3.1.3 Preparation of Protamine Sulfate Fragments

A stock solution of PS fragments was prepared by digesting a PS solution(2 mg/mL in PBS) with Trypsin (200 ng/mL for 60 min at 37° C.). Theenzyme was subsequently inactivated by heat (90° C. for 10 min) followedby ultrafiltration using a 3 kDa membrane (Amicon® Ultra centrifugalfilter). Due to the cut-off of the membrane Trypsin remained in theretentate, whereas the PS fragments were present in the permeate.Complete inactivation of the enzyme was evaluated by spiking 500 μg/mLof full length PS into an aliquot of the obtained PS fragment followedby incubation at 37° C. for 18 h. No degradation of full length PS wasobserved indication complete inactivation/removal of Trypsin.Degradation was monitored by PS-SEC HPLC.

3.1.4 DOE Plan

Samples were prepared according to the pipetting scheme as shown inTable 2. NIV Batch JEV11A74 obtained from a commercial production runwas used as starting sample. NIV was diluted 2-fold to DS using PBSbuffer followed by pH adjustment. 5 mL aliquots were removed andadjuvanted with the corresponding Alum lot 4230, 4074 or a 50/50%mixture of both. The final amount of Alum stock (2% Al2O3) added was 500μg/mL Aluminium (0.1% Al2O3). Each formulation (5 mL) was split into twoparts (2×2.5 mL) using Lo-bind Eppendorf tubes. One aliquot was storedat 2-8° C., another aliquot stored at 22±1° C. (Infors HT Incubator)under gentle shaking (20 rpm).

3.2 Inactivated JEV ELISA (polyclonal based)

Desorption of the antigen from Alum and ELISA analysis was carried outusing polyclonal sheep anti JEV antibodies for coating the 96 well ELISAplates as described in Example 4.

3.3 Inactivated JEV ELISA (Monoclonal Based)

A monoclonal (mAb) based JEV ELISA was developed. The assay is primarilybased on the “polyclonal JEV ELISA” assay format, only a monoclonalanti-JEV antibody (clone 52-2-5) is used for coating. The employed mab52-2-5 was shown to be specific for JEV and to recognize a neutralizingepitope. Mab clone 52-2-5 was obtained by subcutaneously immunizingBALB/c mice with commercially available vaccine lot JEV08J14B. Spleencells of the mice were fused to myeloma cells. From resulting hybridomacells single clones were selected and sub-cloned. The clones werenegatively screened against Bovine Serum Albumin, Protamine sulphate andan extract of the production cell line of the JE-vaccine (Vero cells). Apositive screen was done against Neutralized Inactivated Virus (NW) ofvaccine lot JEV08M20. For screening, microtiter plates were coated withthe relevant antigen and reacted with supernatant of cultures of theselected clones. For detection a goat anti mouse polyclonal antibodyconjugated with alkaline phosphatase was used. Mab clone 52-2-5 wasshown to recognize a neutralizing epitope on domain III of the envelope(E) protein of JEV containing Ser331 and Asp332 (Lin C.-W. and Wu W.-C.J Virol. 2003; 77(4):2600-6). Binding of the mab to the indicatedneutralizing epitope is for instance determined as described in Lin andWu (2003) by site-directed mutagenesis of the domain III at position 331(for instance: S→R), and/or by alanine mutations at or near position 331of domain III, for instance of residues Ser 331 and Asp332, followed byimmunoblots to determine binding of the mab to the mutated proteins.Negative binding results indicate that the epitope of the mab is theneutralizing epitope identified by Lin and Wu (2003). The neutralizingcharacteristic of the epitope gives rise to the assumption that theepitope might be of importance for the antigen to elicit a protectiveimmune response.

JEV samples were analyzed by both ELISA assays, polyclonal andmonoclonal. The relative specific epitope content can be expressed asthe ratio of the total antigen content determined by “monoclonal ELISA”(clone 52-2-5) divided by total antigen content determined by“polyclonal ELISA”. Any differences in the ratio may indicatedifferences in specific epitope content 52-2-5. Results close to 1 wouldcorrespond to high epitope contents, and results close to 0 correspondto low relative epitope content. A low ratio indicates presence ofstructural changes of the neutralizing epitope.

In the course of development of this “mAb ELISA”, differences betweenvaccines lots were detected, which could be correlated with potencyresults of these lots.

3.4 Protamine Sulfate SEC-HPLC

PS (full length) and its fragments were analyzed by size-exclusion HPLC(SEC-HPLC) using a Superdex Peptide 10/300 GL, 10×300 mm, 13 μm (GEHealthcare) using 0.1% (v/v) Trifluoroacetic acid (TFA) in 30%acetinitrile (CAN) as mobile phase at a flow rate of 0.6 mL/min. PScontaining samples were prepared in duplicated, i.e. diluted with mobilephase before injection.

4 Results 4.1 Analysis of Stopper Leachables Used for SpikingExperiments

RP-HPLC elution profiles of concentrated stock solution obtained afterextraction of stoppers under heat compared to FVL SN is shown in FIG. 1.Similar peak pattern as observed for both samples. Due to the harshextraction conditions additional peaks were detected in the concentratethat were not present in FVL samples or present only at a much lowerrelative content Therefore the spiked formulations represent a “worstcase” with regard to leachables and extractables. The total relativecontent of individual peaks in extract concentrate and spikedformulation in comparison to FVL SN is summarized in Table 3. The totalamount of leachables in the stock solution was calculated as the sum ofall peaks detected and expressed in arbitrary units as 67 U/mL. Sincethe stock solution was diluted 16 times into the respective formulation,the resulting total content of leachables was estimated as 4.2 U/mL.This corresponds on average 1.4 fold increase compared to FVL JEV09L37supernatant (3.0 U/mL).

4.2 Analysis of Protamine Sulphate Fragments

PS fragments obtained after cleavage of full length PS by Trypsin areshown in FIG. 2. Similar peak profiles of Trypsin treated PS and alreadydegraded PS present in NIV11A74 were obtained by HPLC (see FIG. 3).

4.3 DOE Evaluation

Formulations prepared for this DOE were analyzed after 4 weeks and 8weeks of incubation at accelerated conditions (22° C.). It wasconsidered that any degradation reaction would be accelerated whenstored at higher temperature compared to normal storage conditions (2-8°C.). However, samples are still stored at 2-8° C. and will be analyzedon a later time point (˜4-6 month). First analysis of samples stored at22° C. for 4 and 8 weeks are shown in the Table 4.

4.3.1 DOE Evaluation after 4 Weeks at 22° C.

Statistical evaluation of the DOE matrix results obtained after 4 weeksat 22° C. showed that the specific epitope content 52-2-5 (expressed asthe ratio of desorbed antigen analyzed by monoclonal/polyclonal ELSIA)was statistically significant influenced (95% confidence level, seeTable 5) by the following factors:

-   -   lower specific epitope content 52-2-5 in presence of Alum lot        4230    -   lower specific epitope content 52-2-5 at lower pH 7    -   Higher specific epitope content 52-2-5 at increased        concentration of formaldehyde

Presence of higher concentration of PS fragments and chlorobutyl rubberleachables did not show any influence on specific epitope content. No2nd or higher order interactions between individual parameters weredetected.

The ANOVA table partitions the variability in “Ratio 4 weeks” intoseparate pieces for each of the effects. It then tests the statisticalsignificance of each effect by comparing the mean square against anestimate of the experimental error. In this case, 3 effects (Alum, pH,Formaldehyde) have P-values less than 0.05, indicating that they aresignificantly different from zero at the 95.0% confidence level. TheR-Squared statistic indicates that the model as fitted explains 74.85%of the variability in Ratio 4 weeks. The adjusted R-squared statistic,which is more suitable for comparing models with different numbers ofindependent variables, is 51.27%. The standard error of the estimateshows the standard deviation of the residuals to be 0.063. The meanabsolute error (MAE) of 0.0353 is the average value of the residuals.The Durbin-Watson (DW) statistic tests the residuals to determine ifthere is any significant correlation based on the order in which theyoccur in your data file. Since the DW value is greater than 1.4, thereis probably not any serious autocorrelation in the residuals. Effectsare also displayed by standardized Pareto chart and main effect plots asshown in FIG. 4.

4.3.2 DOE Evaluation after 8 Weeks at 22° C.

Statistical evaluation of the DOE matrix results obtained after 8 weeksat 22° C. were similar to results obtained after 4 weeks. Evaluationshows that the specific epitope content 52-2-5 (expressed as the ratioof desorbed antigen analyzed by monoclonal/polyclonal ELSIA) wasstatistically significant influenced (95% confidence level, see Table 6)by the following factors:

-   -   lower specific epitope content 52-2-5 in presence of Alum lot        4230    -   lower specific epitope content 52-2-5 at lower pH 7

Presence of higher concentration of PS fragments, chlorobutyl rubberleachables and formaldehyde did not show any influence on specificepitope content. Note that P-value for formaldehyde (P=0.08) is quiteclose to be significant. No 2nd or higher order interactions betweenindividual parameters were detected.

The ANOVA table partitions the variability in “Ratio 8 weeks” intoseparate pieces for each of the effects. It then tests the statisticalsignificance of each effect by comparing the mean square against anestimate of the experimental error. In this case, 2 effects (Alum andpH) have P-values less than 0.05, indicating that they are significantlydifferent from zero at the 95.0% confidence level. The R-Squaredstatistic indicates that the model as fitted explains 75.9% of thevariability in “Ratio 8 weeks”. The adjusted R-squared statistic, whichis more suitable for comparing models with different numbers ofindependent variables, is 53.3%. The standard error of the estimateshows the standard deviation of the residuals to be 0.095. The meanabsolute error (MAE) of 0.057 is the average value of the residuals.Effects are also displayed by standardized Pareto chart and main effectplots as shown in FIG. 5.

Regression analysis was also performed to the fitted data and calculatedregression coefficients are shown in Table 7. The regression equation isdisplayed below which has been fitted to the data including pH, Alum andFormaldehyde. The equation of the fitted model is

“Ratio 8weeks”=0.0228125+0.113125*pH−0.00185625*Alum+0.0315625*Formaldehyde

where the values of the variables are specified in their original units,except for the categorical factors which take the values −1 for the lowlevel and +1 for the high level. The contour of the estimated responseand residual plot is shown in FIG. 6 and FIG. 7. The ratio increaseswhen relative Alum content Lot 4230 decreases and pH increases.

Table 8 contains information about values of “Ratio 8 weeks” generatedusing the fitted model. The table includes:

-   -   (1) the observed value of “Ratio 8 weeks”    -   (2) the predicted value of “Ratio 8 weeks” using the fitted        model    -   (3) 95.0% confidence limits for the mean response

As shown the experimental results are well predicted by the regressionmodel.

5 SUMMARY

Out of the parameters tested, Alum lot 4230 was shown to contributesignificantly to antigen degradation as analyzed bymonoclonal/polyclonal ELISA under accelerated conditions (22° C.,testing time points 4 and 8 weeks). DOE results obtained after 4 and 8weeks at 22° C. show that Alum 4230 is the most significant factor withregard to antigen degradation as detected by the ratio ofmonoclonal/polyclonal ELISA. Formulations made with Alum 4074 (muchhigher purity with regard to residual metal ions) show in general muchhigher specific epitope content.

Formalaldehyde and pH also contributed to antigen stability, but to alower extent. The effect of increased antigen stability in samplesformulated with Alum 4230 at higher formaldehyde level was welldemonstrated (e.g. samples #19 and 29). However, influence offormaldehyde was less pronounced after extended storage period (8 weeksat 22° C.).

Better stability of the antigen was observed at pH 8 compared to pH 7.Protamine sulphate and leachables from chlorobutyl rubber stopper didnot contribute to the antigen degradation.

Example 2

In previous studies (see Example 1) Aluminium hydroxide Lot 4230 wasidentified a significant contributing factor to the observed antigendegradation in FVL09L37. In this particular lot of Aluminium hydroxide(Alum), a much higher residual metal ion content was observed comparedto other Alum lots used for formulation of the inactivated JEV antigen.This Example summarizes additional studies carried out to evaluate theinfluence of metal ions on stability of inactivated JEV. Spiking studieswere conducted with the antigen either present in inactivatedneutralized virus (NIV) solution or in drug product (DP) suspensionfollowing formulation of the antigen with Aluminium hydroxide.

1 STUDY DESCRIPTION

It was previously shown that Alum lot 4230 contains much higher levelsof residual metal ion impurities compared to other Alum lots used forformulation of JEV (see also Example 3). Additional studies wereperformed to evaluate the influence of metal ions on the stability andon a potential surface modification of JEV. The inactivated antigen waseither present in neutralized inactivated virus (NW) solution or in drugproduct (DP) suspension following further dilution of NIV andformulation with Aluminium hydroxide. In another set of experiments,different Alum lots covering a broad content range of residual metalions were used and formulated with a single defined NIV lot. All ofthese formulations still contained residual formaldehyde and bisulphiteat representative concentrations compared to commercial product. Stocksolution of metal salts were dissolved in water and spiked to thesamples to the desired final concentration.

2 DEFINITIONS & ABBREVIATIONS AcCN Acetonitrile

ANOVA Analysis of varianceDOE Design of experimentsDP Drug productDS Drug substanceFBV Final bulk vaccine

FVL Final Vaccine lot

GI Gamma irradiatedHPLC High performance liquid chromatographyLSD Fisher's least significant differencemAb Monoclonal antibodyNIV Neutralized inactivated virusPS Protamine sulphate

RP Reversed Phase

SEC Size exclusion chromatography

SN Supernatant

TFA Trifluoroacetic acidw/o without

3 MATERIALS AND METHODS 3.1 Materials

-   -   Iron(II)chloride tetrahydrate (Sigma, Order no. 44939)    -   Iron(III)chloride hexahydrate (Sigma, Order no. 31232)    -   Nickle(II)sulphate hexahydrate (Sigma, Order no. N4882)    -   Cobalt(II)chloride hexahydrate (Sigma, Order no. 31277)    -   Copper(II)chloride dehydrate (Sigma, Order no. 807483)    -   Zinksulphate heptahydrate (Sigma, Order no. 24750)    -   Crom(III)chloride hexahydrate (AlfaAesar, Order no. 42114)    -   Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)        (Sigma, E5134)    -   Aqua bidest. (Fresenius Kabi, Art no. 0712221/01 A)    -   10×PBS (Gibco, Order No. 14200-091)    -   Formaldehyde solution 37% (Merck, Cat No. 1.040031000)    -   Protamine sulphate (Intercell Biomedical Ltd, Batch no. 086056)    -   LoBind Eppis 2 ml (Eppendorf, Cat. No. 0030 108.132)    -   15 ml Falcon tubes (Greiner, Cat. No. 188724)    -   Incubator Infors HT Incubator Multitron Standard (InforsAG)    -   0.2 μm filter Mini Kleenpak 25 mm (Pall)    -   NIV11A74 and Final bulk vaccine (FBV, formulated with Alum        lot 4539) JEV 11D87 from commercial production runs was obtained        from Intercell Biomedical (Livingston, UK) and stored at 2-8° C.        until further processing    -   Stock solutions of metal salts in water (final concentration 1        mM) used for spiking experiments were prepared and stored at        2-8° C. until usage    -   Aluminium hydroxide samples (2% Al2O3, Brenntag Biosector) were        either retain samples obtained from Intercell Biomedical or        purchased directly from Brenntag. Alum samples were stored at        2-8° C. The following Alum lots were used in this study: 4470,        4563, 4621, 3877, 4230 (non-gamma irradiated and gamma        irradiated)

3.2 Preparation of Metal Stock Solutions 3.2.1 Iron(II) Stock Solution

20 mM Iron(II) stock solution was prepared by dissolving 397 mg ofIron(II)chloride tetrahydrate in 100 mL aqua bidest.

3.2.2 Iron(III) Stock Solution

20 mM Iron(III) stock solution was prepared by dissolving 540 mg ofIron(III)chloride hexahydrate in 100 mL of aqua bidest.

3.2.3 Nickle(II) Stock Solution

20 mM Nickle(II) stock solution was prepared by dissolving 525 mg ofNickle(II)sulphate hexahydrate in 100 mL of aqua bidest.

3.2.4 Cobalt(II) Stock Solution

20 mM Cobalt(II) stock solution was prepared by dissolving 476 mg ofCobalt(II)chloride hexahydrate in 100 mL of aqua bidest.

3.2.5 Copper(II) Stock Solution

20 mM Copper(II) stock solution was prepared by dissolving 341 mg ofCopper(II)chloride dihydrate in 100 mL of aqua bidest.

3.2.6 Zink Stock Solution

20 mM Zink stock solution was prepared by dissolving 575 mg ofZinksulphate heptahydrate in 100 mL of aqua bidest.

3.2.7 Crom(III) Stock Solution

20 mM Crom(III) stock solution was prepared by dissolving 533 mg ofCrom(III)chloride hexahydrate in 100 mL of aqua bidest.

3.3 Preparation of Working Solutions

Working solutions of metal ions (1 mM final concentration if nototherwise stated) were prepared by dilution of metal ion stock solutionswith aqua bidest and sterile filtration via 0.2 μm syringe filter.

3.4 Preparation of Formulation

All formulations were prepared under sterile conditions. NIV and FBVobtained from commercial production runs were adjusted to the desired pHand spiked with aliquots of metal stock solution. All samples werestored in plastic tubes if not otherwise stated. In all formulationsusing Alum, the final Al content was 500 μg/mL, corresponding to 0.1%Al2O3. It has to be noted that metal ions, especially iron (II), iron(III) and to a certain extent Cr (III), formed a precipitate with thephosphate ions present in the buffer resulting in partialco-precipitation of the inactivated virus represented by the lowrecovery determined by size-exclusion HPLC (SEC-HPLC).

3.4.1 Experiment 20110913(NIV): NIV Formulation at Different Metal IonConcentration of Ni(II), Cu(II), Cr(III) with or w/o Presence of PSFragments

NIV 11A74 was adjusted to pH 7 and pH 8 followed by spiking of metalions (Ni(II), Cu(II), Cr(III)) at 100/500/1000 ng/mL finalconcentration. All formulations were stored in low-bind Eppendorf tubesat 22° C. Aliquots of all formulation were also prepared in presence ofprotamine sulphate fragments (50 μg/mL). This was done to evaluate forany effect of PS fragments on JEV stability in presence of metals. Thepreparation of Protamine Sulphate (PS) fragments is described inExample 1. Samples were prepared on the same day (see Table 9) andanalyzed three weeks later. All samples were analyzed by SEC-HPLC, butonly samples at pH 8 (#21-40) were analyzed by ELISA.

3.4.2 Experiment 20110913(DP): DP Formulation at Different Metal IonConcentration of Ni(II), Cu(II), Cr(III)

FBV 11D87 (formulated with Alum Lot 4539) was used in this study. FBVwas adjusted to pH 7 and pH 8 and spiked with Ni(II)/Cu(II)/Cr(III) at100, 500 and 1000 ng/mL to evaluate any metal ion concentration/pHdepended effect. Table 10 shows the experimental design of thisexperiment. All formulations were stored in Falcon tubes at 2-8° C. and22° C. Samples stored at 22° C. were analyzed by SEC-HPLC and ELISAafter 5 weeks.

3.4.3 Experiment 20110812-Metal Spiked DP

Final Bulk Vaccine 11D87 (formulated with Alum Lot 4539) was obtainedfrom a commercial production run and used in this study. Residualformalin in DS was analyzed as 28.1 ppm, residual sulphites was 92.2ppm. Actual content in DP can be considered to be in the same range. FBVJEV11D87 was adjusted to pH 7.0/7.4/7.8 and spiked with 500 ng/mL (finalconcentration) of Fe(II), Fe(III), Ni(II), Co(II), Cu(II), Zn(II). Ametal ion mix formulation was also prepared containing all of theindividual metal ions together in solution. Formulations with Cr(III)were prepared later on and Cr(III) was not included in metal ion mix.Control formulations were only adjusted to the desired pH, but notspiked with metals. All formulations (#1-24) were prepared on the sameday and stored in Falcon tubes at 2-8° C. and 22° C.

Additional Cr(III) spiked samples (#25-27) were prepared by takingaliquots of the control samples stored at 2-8° C. and spiked withCr(III) to a final concentration of 500 ng/mL. Formulations were storedat accelerated conditions (22° C.) only. Table 11 shows the experimentalset-up of this experiment. All samples stored at 22° C. were analyzed byELISA (monoclonal and polyclonal) after 4 weeks and 7 weeks.

3.4.4 Experiment 20110819: DP Formulation Using Various Alum Lots

Spiking studies as described above can give first evidence of possibleinstability of the formulated antigen in presence of certain metals, butmight not be completely representative of the real conditions wheremetals present in Aluminium hydroxide are incorporated in thethree-dimensional structure of the gel resulting in different localconcentration and orientation/accessibility. To overcome theselimitations an initial study was started to simulate the realconditions. A single NIV batch (11A74) obtained from a commercialproduction run was formulated with various Alum lots produced byBrenntag covering a broad range of residual metals. 4.75 mL of NIV wasmixed with 0.25 mL Alum (2%) in Falcon tubes. The final Aluminiumhydroxide concentration was 500 μg/mL (=0.1% Al2O3). Formulated vaccinesamples were stored at 2-8° C. and under accelerated conditions at 22°C. All of these Alum lots contained residual metal ions at differentconcentrations. Alum lot 4230 has the highest level for Fe, Cu, Ni and V(see Example 3). Note that metal ion valences cannot be specified byICP-MS. A mixed Alum sample containing equal amounts of 4230 and 4074was also prepared to get an “intermediate” level for Ni(II) and Cu(II).Samples were analyzed after 6 weeks of storage at 22° C. The residualamount of formaldehyde and sulphite estimated by recalculation fromavailable DS analysis results corrected by dilution factor of NIV to DSwas 76 ppm formaldehyde and 192 ppm sulphite respectively.

3.1 Antigen Desorption from Aluminium Hydroxide for SEC-MALLS Analysis

Viral particles were desorbed from Aluminium hydroxide. ˜625 μL of DPwas spun down (8° C., 5 min, 3300×g) and the supernatant was eitherdiscarded if not otherwise stated or analyzed by JEV-SEC-MALLS to detectthe unbound antigen concentration. Viral particles were desorbed bysuspending the Aluminium hydroxide particles with 62.5 μL 0.8 Mpotassium phosphate buffer (pH 8) containing BSA (50 μg/mL). BSA wasadded to the desorption buffer for SEC-MALLS analysis to minimize lossescaused by unspecific adsorption of the antigen. After shaking (500 rpm)the Aluminium hydroxide particles for 10 min at room temperature,particles were removed by centrifugation and the supernatant wascollected into a LoBind Eppendorf tube and the desorption procedurerepeated on remaining sample. The pooled desorbed antigen (˜5×concentrated sample; final volume 125 μL; starting volume ˜625 μL) wasthen further analyzed by SEC-MALLS.

3.2 SEC-MALLS HPLC Method

Desorbed antigen was analyzed by SEC-MALLS. In brief, followingdesorption of the antigen from Aluminium hydroxide 100 μL of the pooleddesorbed material (˜5× concentrated) were subsequently loaded onto aSuperose 6 10/300 GL SEC column. 1×PBS+250 mM NaCl was used as mobilephase. Ultraviolet (UV) 214 nm and MALLS signals of viral particles wererecorded and analysed using Chromeleon and ASTRA software packages.

3.3 Inactivated JEV ELISA (Polyclonal Based)

Desorption of the antigen from Alum and ELISA analysis was carried outusing polyclonal sheep anti JEV antibodies for coating the 96 well ELISAplates as described in Example 4.

3.4 Inactivated JEV ELISA (Monoclonal Based)

During course of this investigational testing, a monoclonal (mAb) basedJEV

ELISA was developed. The assay is primarily based on the “polyclonal JEVELISA” assay format, only a monoclonal anti-JEV antibody (clone 52-2-5)is used for coating and the current polyclonal antibody for detection.The employed mab 52-2-5 was shown to be specific for JEV and torecognize a neutralizing epitope. Mab clone 52-2-5 was obtained bysubcutaneously immunizing BALB/c mice with commercially availablevaccine lot JEV08J14B. Spleen cells of the mice were fused to myelomacells. From resulting hybridoma cells single clones were selected andsub-cloned. The clones were negatively screened against Bovine SerumAlbumin, Protamine sulphate and an extract of the production cell lineof the JE-vaccine (Vero cells). A positive screen was done againstNeutralized Inactivated Virus (NIV) of vaccine lot JEV08M20. Forscreening, microtiter plates were coated with the relevant antigen andreacted with supernatant of cultures of the selected clones. Fordetection a goat anti mouse polyclonal antibody conjugated with alkalinephosphatase was used. Mab clone 52-2-5 was shown to recognize aneutralizing epitope on domain III of the envelope (E) protein of JEVcontaining Ser331 and Asp332 (Lin C.-W. and Wu W.-C. J Virol. 2003;77(4):2600-6). Binding of the mab to the indicated neutralizing epitopeis for instance determined as described in Lin and Wu (2003) bysite-directed mutagenesis of the domain III at position 331 (forinstance: S→R), and/or by alanine mutations at or near position 331 ofdomain III, for instance of residues Ser 331 and Asp332, followed byimmunoblots to determine binding of the mab to the mutated proteins.Negative binding results indicate that the epitope of the mab is theneutralizing epitope identified by Lin and Wu (2003). The neutralizingcharacteristic of the epitope gives rise to the assumption that theepitope might be of importance for the antigen to elicit a protectiveimmune response.

JEV samples were analyzed by both ELISA assays, polyclonal andmonoclonal. The relative specific epitope content can be expressed asthe ratio of the total antigen content determined by “monoclonal ELISA”(clone 52-2-5) divided by total antigen content determined by“polyclonal ELISA”. Any differences in the ratio may indicatedifferences in specific epitope content 52-2-5. Results close to 1 wouldcorrespond to high epitope contents, and results close to 0 correspondto low relative epitope content. A low ratio indicates presence ofstructural changes of the neutralizing epitope.

In the course of development of this “mAb ELISA”, differences betweenvaccines lots were detected, which could be correlated with potencyresults of these lots.

3.5 Statistical Evaluation

Statistical evaluation was done with Statgraphic Plus 3.0.

4 RESULTS

4.1 Experiment 20110913(NIV): NIV Formulation at Different Metal IonConcentration of Ni(II), Cu(II), Cr(III) with or w/o Presence of PSFragments

SEC-HPLC results of NIV formulations (pH 7 and pH 8) containing metalions [Ni(II), Cu(II), Cr(III)] w/and w/o PS fragments are summarized inTable 12. SEC-HPLC results show that antigen recoveries of most of thesamples was >80%. Some samples (#7, #36, #38) showed slightly reducedrecoveries in the range of 70-80%. It has to be noted that the actualvirus content is quite low and precision of HPLC results can beestimated as approx. ±20%. Since for samples #36 and #38 the recoveriesfor following formulations (#37, #39) at next level of individual metalion content were higher again, these differences might be caused byassay variability and were not considered as significant. Based on theresults obtained it was not possible to clarify the influence of metalions with respect to the recovery of soluble inactivated JEV. However,SEC-HPLC only gives information about content of soluble virus, but noinformation about any potential surface modification. Only formulationsprepared at pH 8 were also analyzed by ELISA (duplicate analysis). Theratio of monoclonal/polyclonal ELISA was calculated and can be used forcomparison purpose of results. Analysis of samples by ELISA (see Table13) do not show any significant influence of tested metals ondegradation of inactivated JEV at pH 8 after three weeks at 22° C. Theremight be a trend of decreasing ratio in presence of Cu(II), but overallit appears that an incubation time of three weeks at 22° C. seems not besufficient to detect any significant degradation. As also shown in DOEexperiment (Example 1) inactivated JEV appears to have higher stabilityat pH 8 when stored at accelerated conditions at 22° C. and this wouldalso contribute that significant effects were not observed. In thisexperiment it was also shown that PS fragments do not have any influenceon JEV stability. This is also well in agreement with DOE results. NIVsamples 1-20 formulated at pH 7 showed significant reduction inmonoclonal epitope content in presence of Cu(II). At the highest testedconcentration (1000 ng/mL) the ratio was close to zero indicationsignificant structural changes of the antigen.

4.2 Experiment 20110913(DP): DP Formulation with Different Metal IonConcentration of Ni(II), Cu(II), Cr(III)

Analysis of desorbed JEV antigen is summarized in Table 14 (SEC-HPLC)and Table 15 (ELISA). Antigen recoveries for all samples as determinedby SEC-HPLC was >80% after 5 weeks at 22° C. indicating no significantinfluence of tested metal ions on desorption recovery. As shown in FIG.8, there is a trend of decreasing ratio as analyzed by ELISA in presenceof Cu(II) and Cr(III) at pH 7. Formulations at pH 8 appear to be morestable.

4.3 Experiment 20110812(DP): Metal Ion Spiked DP

In this experiment FBV11D87 was used as starting material. Theformulation pH value was adjusted in a more narrow range (pH 7.0, 7.4,7.8) and additional metal ions were used for spiking, each at 500 ng/mL(final concentration). The metal ion mix contained all individual metalions with the exception of Cr(III) in single formulations (each metal at500 ng/mL). ELISA results obtained after 4 weeks and 7 weeks at 22° C.are summarized in Table 16. Results are also displayed as graphs in FIG.9.

Statistical evaluation of stability samples stored at 22° C. for 7 weekswas performed. ANOVA (analysis of variance) showed significant effectsof parameters (pH and metal type) on antigen stability, that isexpressed as the ratio of monoclonal/polyclonal ELISA (see Table 17).The ANOVA table decomposes the variability of Ratio into contributionsdue to various factors. Since Type III sums of squares have been chosen,the contribution of each factor is measured having removed the effectsof all other factors. The P-values test the statistical significance ofeach of the factors. Since the P-values for pH and metal ion type areless than 0.05, these factors have a statistically significant effect onRatio at the 95.0% confidence level.

In Table 18 a multiple comparison procedure is applied to determine thesignificance of the differences observed with respect to the means.Significant effect on ratio was shown for Cu(II) and the metal mixcompared to the non-spiked control formulations. The bottom half of theoutput shows the estimated difference between each pair of means. Anasterisk has been placed next to 7 pairs, indicating that these pairsshow statistically significant differences at the 95.0% confidencelevel. At the top of the page, 3 homogenous groups are identified usingcolumns of X's. Within each column, the levels containing X's form agroup of means within which there are no statistically significantdifferences. The method currently being used to discriminate among themeans is Fisher's least significant difference (LSD) procedure. Withthis method, there is a 5.0% risk of calling each pair of meanssignificantly different when the actual difference equals 0.

Significant effect on ratio was shown for Cu(II) and the metal ion mix.The influence of other metal ions might become significant at longerstorage period. The metal ion mix contained the highest totalconcentration and might represent a worst case. However, it wasconcluded that several metal ions present in Alum might contribute todegradation, each to different extent. These results further support theproposed root cause of metal ion-catalysed antigen degradation. It hasto be noted that spiking experiment might not fully simulate the realconditions of residual metal ion impurities present in Alum 4230. Metalions are incorporated in the Alum structure and the local concentrationand orientation might be different from metal ions used in spikingexperiments. It is also known that metal ions (e.g. Fe) have lowsolubility in presence of phosphate ion (PO⁴³⁻). Therefore the actualconcentration of soluble metal ions and contribution of metals presentas metal-phosphate complex on JEV degradation is unknown.

4.4 Experiment 20110819: Preparation of DP Samples with Different AlumLots

Spiking studies as described earlier can give first evidence of possibleinstability of the formulated antigen in the presence of some metalions, but might not be completely representative of the real conditionswhere these metal ions present in Aluminium hydroxide are expected to beincorporated in the three-dimensional structure of theAluminium-hydroxide gel resulting in different local concentration andorientation/accessibility. To overcome these limitations an initialstudy was started to simulate the real conditions. A single NIV (11A74)was formulated with various Alum lots obtained by Brenntag covering abroad range of residual metals. Formulated vaccine samples were storedat 2-8° C. and under accelerated conditions at 22° C. All of these Alumlots contained residual metal ions at different levels. Lot 4230 had thehighest level for Fe, Cu, Ni and V (see Table 19). Note that the actualcontent of metal ions present in the formulated product is only 1/20 ofthe concentration in Alum (2%) stock solution. Note that metal ionvalences cannot be specified by ICP-MS. Analysis of desorbed antigen byELISA of samples stored at 22° C. for 6 weeks is shown in Table 20.

Pooled standard deviation was calculated from all samples(spooled˜0.075) as a measurement of experimental uncertainty. Meanvalues for ratio and 95% confidence intervals (calculated based onspooled) were plotted against the individual formulations (see FIG. 10).Samples formulated with Alum 4230 showed a trend to lower ratio comparedto the other samples. However, differences were not as large to showstatistical significant differences between the various formulations.

5 SUMMARY

It was shown that metal ions contribute to the degradation of theinactivated JEV under accelerated storage conditions (22°). In spikingstudies higher concentration of residual metal ions (range 100-1000ng/mL) were used than present in FVL formulated with Alum lot 4230 (e.g.Fe˜310 ng/mL; Cr˜64 ng/mL; Ni˜52 ng/mL). Cu content in FVL can be onlyestimated as 3 ng/mL based on ICP-MS data of 2% Alum stock solutionsince LOD is 25 ng/mL. Higher metal concentration and storagetemperature were chosen to increase the rate of any potentialdegradation reaction. In fact, for FVL JEV09L37, the potency lossoccurred after 11 month stored at 2-8° C. It was also shown that metalscan form insoluble complexes with phosphate ions making estimation ofactual levels of metals present difficult. In spiking experiments ELISAresults showed statistically significant structural changes of virussurface in as little as 4 weeks at 22° C. in presence of metal ions. Itwas shown that the ELISA ratios for formulations containing Cu(II) andmetal ion mix (containing Fe(II), Fe(III), Co(II), Cu(II) and Zn(II))were statistically significant lower compared to the non-spiked controlformulation. Cu(II) was also found in Alum lot 4230 (2% stock solution)at 64 ng/mL, corresponding to ˜3 ng/mL in FVL. In all other Alum (2%)lots Cu(II) content was <25 ng/mL (below limit of detection).

For formulation experiments of the antigen using different Alum lots,longer storage time (>6 weeks at 22° C.) at accelerated conditions isrequired. There is a trend that formulations prepared with Alum 4230showed lower ELISA ratios compared to other lots. Slower degradationrate compared to spiked formulation might be contributing to lower metalion content in commercial Alum lots. It was also observed that theantigen shows higher stability at pH 7.5-8 compared to pH 7 and that PSfragments do not contribute to any degradation reaction. These resultsare in good agreement with DOE results described in Example 1.

Example 3

As part of the out-of-specification investigation concerning FVLJEV09L37, the used Aluminum hydroxide lot (lot 4230) was determined tobe the most probable root cause for the observed loss of potency.Aluminum hydroxide (referred to as Alum during the manufacturing processof JE-PIV) is purchased from Brenntag Biosector as autoclaved suspensiontermed “ALHYDROGEL® Aluminium Hydroxide Gel Adjuvant”. Each batch issterilized by radiation prior to use in the JEV production process.

A number of different ALHYDROGEL® batches were analyzed for appearance,metal ion content and physical properties.

1 Introduction 1.1 Aluminum Hydroxide

Brenntag Biosector's ALHYDROGEL® has a specified Aluminum content of 10mg/mL which translates to 2% Al₂O₃ and 3% Al(OH)3, respectively. Furtherspecifications are Nitrogen (max 0.005%), free Sulphate (max 0.05%),total Sulphate (max 0.1%) and pH (6.5±0.5). it has a shelf life of 26months when stored at room temperature.

1.2 Generation of Aluminum Hydroxide

ALHYDROGEL® 2% (referred to as Aluminum hydroxide) is manufactured byBrenntag (CAS no. 21645-51-2).

1.3 Use of Aluminum Hydroxide Lots in JEV Manufacturing

For the production of commercial JEV vaccine batches a total of 5different Brenntag ALHYDROGEL® 2% lots have been used so far.

2 Definitions & Abbreviations

ALHYDROGEL® 2% Aluminum hydroxide solution (also referred to as alum)

DS/DP Drug Substance/Drug Product ESG Environmental Scientifics GroupF-AAS Flame Atomic Absorption Spectrometry FVL Final Vaccine Lot GF-AASGraphite Furnace Atomic Absorption Spectrometry ICP-MSInductively-Coupled-Plasma Mass Spectrometry JEV Japan EncephalitisVirus JE-PIV Japan Encephalitis Purified Inactivated Virus LOQ Limit OfQuantification P&TDPatch & Technical Development PSD Particle SizeDistribution PZC Point of Zero Charge QCI Quality Control Immunology 3Materials and Methods 3.1 ALHYDROGEL® Batches

ALHYDROGEL® 2% lots: 3877, 4074, 4187, 4230, 4414, 4470, 4539, 4563,4587, 4621 (not all Alum lots listed were used in the formulation ofJE-PIV) ALHYDROGEL® 2% 7× washed lots: 4577, 4580, 4596 (sourced fromBrenntag, not typical of the 2% Alum received for formulation)

3.2 ALHYDROGEL® PSD Measurements

Aluminum hydroxide particle size distribution (PSD) was analyzed on aMalvern Mastersizer 2000 μP system with a 20 mL sample cell. ALHYDROGEL®2% bulk substance was diluted 1:20 in water and 1 mL was added to thesample cell. Final dilution of sample in sample cell was therefore 400fold (0.005% Aluminum hydroxide).

3.3 ALHYDROGEL® Zeta-Potential Measurements

Zetapotential and point of zero charge (PZC) was measured on a MalvernZetasizer ZS system equipped with a MPT-2 autotitrator. ALHYDROGEL® 2%bulk substance was diluted 1:20 in PBS and equilibrated over night atroom temperature. For recording of the charge titration curve the pH wasadjusted using 100 mM HCl and 100 mM NaOH solutions. PZC was determinedby extrapolation of the zero charge in the titration plot (intersectionof titration curve and x-axis). Point of zero charge corresponds to thepH value where the surface of the sample has no net charge.

3.4 Analysis of Metal Ions in Aluminum Hydroxide

Selected metal ions were analyzed either by inductively-coupled-plasmamass spectrometry (ICP-MS), flame atomic absorption spectrometry (F-AAS)and graphite furnace atomic absorption spectrometry (GF-AAS) at theMedical Laboratory Bremen (Germany). In short, samples containingAluminum hydroxide were treated with conc. HNO3 under heat until a clearsolution is obtained. The clear solution is then further diluted andanalyzed. The presence and content of following metal ions weredetermined: Pb, Cd, Cr, Co, Fe, Cu, Ni, Ag, W and Al. Depending on thesample dilution, the limit of quantification (LOQ) was 5 to 25 ng/mL.

In addition a semi-quantitative 70-Element scan was performed by ESG(UK) using a combination of ICP-MS (Agilent 7500ce) and ICP-AES (PerkinElmer Optima 4300DV), which were calibrated using certified standards.The element scan is a screening method and not as sensitive as tracemetal analysis for selected metals as performed by Medical LaboratoryBremen. However, such a screening gives a good overview about thepresence and levels of certain metals.

4 Results 4.1 Determination of ALHYDROGEL® Particle Size Distribution

Table 21 summarizes PSD data of two sublots each of ALHYDROGEL® lots4230 and 4740. Distribution results are shown in FIG. 11. Mean particlewas ˜2-4 μm with populations of smaller (<1 μm) and larger (>20 μm)particles being present in all four samples. The four tested ALHYDROGEL®samples showed no significant difference in mean particle sizedistribution.

4.2 Zeta-Potential Measurements

Two sublots each of ALHYDROGEL® lots 4230 and 4740 (2% stock solutiondiluted 20 fold in PBS and equilibrated overnight at RT before analysis)were analyzed for point of zero charge. Table 22 summarizes results ofPZC for the four samples showing very similar PZC in PBS buffer.Titration curves are shown in FIG. 12. No difference in the titrationcurves and PZC could be observed between the four analyzed samples.

4.3 Determination of Residual Metal Ion Content in ALHYDROGEL® Batches

The current limits for Fe in 2% Aluminum hydroxide solutions accordingto the Ph. Eur. are 15 ppm (=15 μg/mL) and a total maximum of 20 ppm(=20 μg/mL) for other heavy metals (such as Pb). However, aconcentration of 15 ppm Fe would correspond to 0.27 mM Fe in solution.Taking into consideration that even trace amounts of residual metal ionscan catalyze a variety of degradation reactions for proteins (e.g.oxidation, activation of proteases etc.) and that metals remain stablein solution, differences in metal ion content between Aluminum hydroxidelots might cause differences in antigen stability over time.

The concentrations of a number of metal ions in commercially availablealuminum hydroxide lots were analyzed using ICP-MS. The results of theseanalyses are summarized in Table 23. Lots 4074, 4230, 4470, 4414 and4539 were used in the production of commercial JEV batches. As a 2%ALHYDROGEL® stock solution equals an Al concentration of 10 mg/mL thequantification of Al content in the different samples can be used asreference for the results obtained for the other metal ions. Indeed anaverage Aluminum content of 10.3 mg/mL could be measured showing theaccuracy and reproducibility of the method.

When comparing the different ALHYDROGEL® lots large variations in theamount of contaminating metal ions could be observed. Most notablecontaminating metal ions are Fe, Cr and Ni which were detected in allbatches. In addition lot 4230 contained detectable amounts of Cu whichwas below LOQ in all other batches.

However, it has to be noted that none of these metals were detected inquantities near the specifications of ALHYDROGEL® mentioned above. Forexample the highest concentration of iron found in lot 4230 was 5.6μg/mL or roughly 40% of the permitted concentration.

An “improved” ALHYDROGEL® is washed 7 times with water during thepurification step instead of only 4 times for standard ALHYDROGEL®. Totest if this additional washing steps would result in reduced metal ioncontamination three different lots (4580, 4596 and 4577) were analyzed.Results are included in Table 23. No difference in metal ions comparingto the standard grade ALHYDROGEL® could be observed suggesting that themetal ions are either strongly bound to the surface of the Aluminumhydroxide particles or actually co-precipitate during the productionprocess.

FIG. 13 shows a comparison of the different ALHYDROGEL® lots analyzed.The total contaminating metal ion content for all tested contaminatingelements are shown with the absolute shares for the three major metalsFe, Cr and Ni depicted in different colors. As can be seen lot 4074 hasvery little contaminating metal ions compared to the majority of theother analyzed batches. Only lot 3877 showed a similar lot contaminationwhereas lot 4230 shows by far the highest contamination of all batchesanalyzed during this investigation.

During the investigational testing a large variation in the metal ioncontent was observed between different batches of ALHYDROGEL® (see FIG.13). To test if these contaminating metal ions are located in theAluminum hydroxide fraction or in the supernatant Lot 4230 was separatedinto a supernatant and a sediment fraction (see Table 24). As can beseen less than 2% of the metal ions could be detected in the supernatantindicating that all contaminating metal ions are either bound to theAluminum hydroxide particle surface or inside the particle structures.The local metal ion concentration can therefore be estimated to be atleast 50-100 times higher since the solid volume fraction (volume ofAlum-pellet after centrifugation) of 0.1% Al2O3 (corresponding 0.5 mg/mLAl) used in JEV vaccine formulation is approx. 10-20 μL per 1000 μL ofFVL.

5 Summary

ALHYDROGEL® is used in a 0.1% final concentration as adjuvant in thecurrent JEV vaccine formulation. During the investigation of anout-of-specification (OOS) potency result for production FVL JEV09L37 anevaluation of the ALHYDROGEL® production process was initiated. A totalof 13 different ALHYDROGEL® lots were analyzed for the presence ofcontaminating metal ions that could reduce protein stability.

Large variations in the concentration of a number of metal ions wereobserved for different ALHYDROGEL® lots. When analyzing the rawmaterials it was shown that these contaminations were present at thesame concentration as found within the ALHYDROGEL®.

Higher levels of Fe, Ni and Cu ions were noted in ALHYDROGEL® lot 4230when compared to the other investigated lots. Lot 4230 was the only onewhere residual Cu ions were detected. This lot 4230 was used for theformulation of FVL JEV09L37.

When analyzing supernatant and insoluble fraction of an ALHYDROGEL®batch these contaminating metal ions could only be found in theprecipitate indicating that these ions are either bound to the Aluminumhydroxide particle surface or actually part of the particle.

Although macroscopic and in composition different from other ALHYDROGEL®lots used for JEV production, lot 4230 fulfilled all requirementsdetailed by the Ph. Eur. Also physical characterization (particle sizedistribution and point of zero charge) showed no differences between lot4230 and other ALHYDROGEL® lots not showing these high metal ioncontaminations.

Example 4 1.1. Materials, Equipment and Methods 1.2. Equipment

Analytical balance (readability of 0.1 mg; e.g. Mettler ToledoXP205DR/M)Precision balance (readability of 0.1 g; e.g. Mettler Toledo, Model NoXS6002S Delta Range)Filter Units 0.22 μm (e.g. Stericup Cat No SCGPV01RE) or 0.2 μm filtersystem (e.g. 50 mL Millipore Steriflip)

Freezer (−20° C.) and Ultra-Freezer (−80° C.) Fridge (+2 to 8° C.)

Magnetic stirrer (e.g. KIKA Labortechnik RCT basic) and magnetic stirbarsMicroplate Washer: e.g. BioTek ELx405Microplate Reader: e.g. BioTek Synergy 2 and Gen5 Secure software

Microplate Incubator (37° C.)

Microtiter Sealing tape (e.g. Thermo Electron 9503130)Multichannel pipettes and tips (e.g. Eppendorf Research Pro 50-1200 μL,Eppendorf Research, 10-100 μL)pH meter (e.g. WTW Series ino Lab, Terminal 740 and pH/Cond. 740)Pipettes and tips (e.g. Eppendorf Research, 0.5-10 μL, 2-20 μL, 20-200μL, 100-1000 μL, 500-5000 μL)Pipettor (e.g. IBS Biosciences Pipetboy)PP Tubes 15 mL (e.g. Sarstedt 62.515.006) or PP tubes 50 mL (e.g.Greiner 227261)Reagent Reservoir 50 mL (e.g. Corning Incorporated 4870)Serological pipettes (e.g. Falcon, 2 mL, 5 mL, 10 mL, 25 mL, 50 mL)

Titertube Micro Tubes—Bulk (BioRad 223-9391)

Vortex mixer (e.g. VWR Analog Vortex Mixer, Model No 945304)1.5 mL or 2.0 mL Eppendorf LoBind tubes (Cat No 0030 108.116, Cat No0030 108.132, respectively)96 well Microplate (F96 Cert. Maxisorp Nunc-Immunoplates)

For Analysis of DP Samples in Addition:

Bench top centrifuge (e.g. Beckman coulter, Microfuge 16 Centrifuge, CatNo A46473)Orbital shaker (e.g. Eppendorfer Thermomixer compact)50 mL PP tubes (e.g. Greiner 227261)

1.3. Reagents

PBS 10× (e.g. Gibco, Cat. No 14200-083)Tween 20 (e.g. Sigma Cat. No P7949)2M Sulphuric Acid (Volumetric solution, e.g. Fisher, Cat. No. J/8410/17)De-ionised water, e.g. (Milli-Q, 18.2Ω)Sodium carbonate—bicarbonate capsules (e.g. Sigma, Cat. No. C3041)Hydrochloric acid (HCl) 1 mol/L (e.g. Merck, Cat. No 1.09057.1000)Sodium hydroxide (NaOH) 1 mol/L (e.g. Merck, Cat. No 1.09132.1000)Glycerol (e.g. Sigma)

For Analysis of DP Samples in Addition:

Di-potassium hydrogen phosphate trihydrate (e.g. Sigma, Cat No. P5504)Potassium di-hydrogen phosphate (e.g. VWR, AnalaR Normapur, Cat No.26936.260)Albumin, Bovine Serum (BSA), ELISA grade (e.g. Sigma, Cat. No. A3059)TMB Substrate (e.g. BioFX, TMBW-1000-01)Donkey anti rabbit IgG HRP Conjugate (Jackson Immuno Research, Cat No711-035-152) Reconstitution:The content of 1 vial (0.4 mg) is reconstituted in 0.5 mL of de-ionisedwater and thoroughly mixed until total dissolution. Add 0.5 mL ofGlycerol and mix it further until homogeneity. Aliquots are stored at−20° C. until use.

Inactivated JEV Reference Standard (Intercell Biomedical Ltd.)

Purified sheep anti-JEV (Intercell Biomedical Ltd.)Purified rabbit anti-JEV (Intercell Biomedical Ltd.)

1.4. Solutions

a) 0.05M carbonate buffer at pH 9.6 (used for coating of ELISA plates)

For 100 mL buffer, dissolve one bi-carbonate/carbonate buffer capsule in100 mL de-ionised water. Check the pH and adjust to 9.6±0.1 with HCl orNaOH if required. Use on the day of preparation only. Keep ELISA coatingbuffer at RT during the day of use, then discard.

b) ELISA wash buffer and part of block/sample diluent (PBS-T)

Prepare approximately 1 litre for every plate used. Dilute 10×PBS stock1+9 in de-ionised water, mix well and check pH (7.4+/−0.1), adjust with1M HCl or 1M NaOH as required. Add 0.05% (v/v) TWEEN® 20, mix well.

e.g. ELISA wash buffer (PBS-T) [1 L]:

100 mL 10x PBS 900 mL de-ionised water

Mix well, check/adjust pH (7.4+/−0.1).

0.5 mL TWEEN® 20

Mix well.

Use on the day of preparation only; keep ELISA wash buffer at RT duringthe day of use, then discard.

c) Blocking solution: 5% BSA in PBS-T

Prepare approximately 25 mL for every plate. Measure required quantityof PBS-T into a clean glass bottle using a serological pipette. Add aclean magnetic stir bar. Weigh the required amount of BSA, add to thesurface of the PBS-T and mix gently on a magnetic stirrer until all theBSA has gone into solution. Filter solution using a 0.2 μm filter(either Steriflip filter system or syringe filter).

e.g. Blocking solution [100 mL]

5 g BSA 100 mL PBS-T

Use on the day of preparation only; keep blocking solution at RT duringthe day of use then discard.

d) Sample diluent: 1% BSA in PBS-T

Prepare as above but using 1 g of BSA per 100 mL PBS-T, approximately 25mL is required per plate.

e.g. Sample diluent [100 mL]

1 g BSA 100 mL PBS-T

Use on the day of preparation only; keep sample diluent at RT during theday of use, then discard.

For Analysis of DP Samples in Addition:

e) 1×PBS

Prepare 1 part 10×PBS with 9 parts de-ionised water

e.g. 1×PBS [100 mL]

10 mL 10×PBS

90 mL de-ionised water

Use on the day of preparation only; keep 1×PBS at RT during the day ofuse, then discard.

f) 20×ELISA buffer

Weigh an appropriate amount of BSA into a suitable container to make a20× solution. Add the appropriate volume of 1×PBS. Add TWEEN® 20 to afinal concentration of 0.05% Mix on the magnetic stirrer until the BSAis fully dissolved. Filter the solution through a 0.2 μm filter (usingeither STERIFLIP filter system or syringe filter) into a sterilecontainer (and aliquoted as needed).

e.g. 20×ELISA Buffer [25 mL]

5 g BSA 25 mL 1xPBS 12.5 μL TWEEN ® 20

The solution can be stored at +2-8° C. for 1 week.

g) 2×ELISA buffer

It is prepared by dilution of the 20×ELISA Buffer with 1×PBS (1 part 20×ELISA Buffer and 9 part 1×PBS).

e.g. 2×ELISA Buffer [20 mL]

2 mL 20×ELISA Buffer

18 mL 1×PBS

Use on the day of preparation only; keep 2×ELISA buffer at RT during theday of use, then discard.

h) Desorption buffer

-   -   Potassium phosphate stock solution: Make a 3× stock solution of        potassium phosphate (2.4M) by dissolving the appropriate volume        of di-potassium phosphate trihydrate and of potassium dihydrogen        phosphate in de-ionised water. Place on a magnetic stirrer and        once dissolved make up the required volume, check that the pH of        the solution is 8.0+/−0.1. Filter through a 0.2 μm filter.

e.g. 3× stock solution of Potassium phosphate (2.4M) [50 mL]

23.963 g Di-potassium phosphate trihydrate  2.041 g Potassium dihydrogenphosphate

Make up to 50 mL De-ionised water

Store at +2°−8° C. for up to 1 month.

Make working strength desorption buffer (0.8M potassium phosphate buffercontaining 1% BSA and 0.05% TWEEN® 20) by adding the appropriate volumeof potassium phosphate stock (2.4M), of TWEEN® 20 and of BSA to therequired volume of de-ionised water. Mix thoroughly and use on the dayof preparation.

e.g. working strength desorption buffer [15 mL]

5 mL Potassium Phosphate (2.4M) 7.5 μL TWEEN ® 20 0.15 g BSA 10 mLDe-ionised water

Keep working strength desorption buffer at RT during the day of use.

1.5. Test Samples and Antibodies Test Samples:

-   -   Drug Substance and/or NIV (various batches)    -   JEV Vaccine samples (final bulk vaccine and final vaccine lot)

Inactivated JEV Reference Standard (Neutralised Inactivated Virus—NIV)(Intercell Biomedical Ltd.)

Polyclonal Antibodies:

-   -   Coating antibody: Purified Sheep anti-JEV (Intercell Biomedical        Ltd.)    -   Primary detection antibody: Purified Rabbit anti-JEV (Intercell        Biomedical Ltd.)

Secondary conjugated antibody: Donkey anti-Rabbit HRP Conjugate (JacksonImmuno Research Cat. No 711-035-152)

2 Procedure 2.1. Plate Coating

-   -   Label the plate with plate number, date and analyst.    -   Prepare fresh 0.05M carbonate buffer (pH 9.6) on the day of        plate coating. Allow approximately 12 mL for each plate coated.    -   Remove the required number of aliquots of the coating antibody        from the freezer and allow thawing at RT. Prepare a dilution of        Purified Sheep anti-JEV in carbonate buffer. Mix well by        inversion of the tube.    -   Using the multichannel pipette, apply 100 μL/well to a 96-well        Maxisorp plate within 15 min of preparation of the antibody        dilution.    -   Cover with microtiter sealing tape and incubate 17 to 72 hrs at        +2-8° C.

2.2. Washing

-   -   Remove plate from the refrigerator and allow warming to room        temperature.    -   Wash the plate/s with the Microtiter plate washer 3 times using        the respective wash program (300 μL per well, three times, final        dispense). After that: remove any remaining wash buffer by        decanting. Invert the plate and blot it against a clean paper        towel. Do not allow microtiter plate to dry between wash steps        and reagent addition.

2.3. Blocking

-   -   Prepare a Blocking Solution 5% (w/v) of BSA in PBS-T as above.    -   Apply 200 μL Blocking Solution per well, cover the plate(s) with        a cover plate and incubate at 37° C. for 1 hour+/−10 min.

2.4. Preparation of Standard Curve Dilutions

-   -   Remove the NIV reference standard from the freezer, allow        thawing at RT, mix well. Prepare a 1 AU/mL stock dilution of the        current reference standard; use at least 20 μL of NIV reference        standard for dilution.

e.g. NIV reference standard Pre-dilution:

Concentration: 235 AU/mL (lot No 03/2009)

To prepare a 1 AU/mL working standard solution dilute it 1 to 235 insample diluent:

4680 μL sample diluent  20 μL NIV reference standard

-   -   Prepare then the following working standard solutions from the 1        AU/mL pre-dilution:        0.8 AU/mL, 0.6 AU/mL, 0.4 AU/mL, 0.2 AU/mL, 0.1 AU/mL and 0.05        AU/mL in sample diluent.

2.5. Quality Control Samples

a) Quality control (QC) samples (for example at 0.75, 0.30 and 0.18AU/mL) should be made from the NIV reference standard pre-dilutionfreshly at the time of the assay then discarded once used.

b) These controls are part of the system suitability criteria and allowthe performance of the assay to be monitored over time.

2.6. Preparation of Test Samples Drug Substance Preparation

Drug substance test samples are received for testing at unknownconcentrations. These will be tested at six dilutions in triplicate. Thedilutions will be made independently into the range of the standardcurve, e.g. pre-dilution of 1 in 15 or other suitable dilution then sixdilutions with sample buffer.

NIV Sample Preparation

NIV samples will be received for testing at unknown concentrations andpre-diluted in the range of the standard curve (e.g. 1 in 30 or othersuitable dilution) then diluted six times in the same manner as the DSsamples.

Drug Product Supernatant Preparation

a) For the analysis of Bulk-DP samples mix well sample by vortexing.

Transfer exactly 1 mL into a 1.5 mL LoBind Eppendorf tube.

For the analysis of final product container samples transfer the contentof 2 syringes (0.6 mL per syringe) of the same lot into a 1.5 mL LoBindEppendorf Tube. Mix content of tube thoroughly by inversion to ensurehomogeneity of the DP and transfer exactly 1 mL into fresh 1.5 mL LoBindEppendorf tube.

b) Centrifuge tubes containing 1 mL DP each at 3300×g for 5 minutes.

c) For each sample, pipette 25 μL of 20×ELISA buffer into fresh LoBindEppendorf tube.

d) Carefully remove 475 μL of the supernatant without disturbing thealum pellet and transfer into the tube containing the 20×ELISA buffer.Mix gently by inversion. Re-spin 2 min at 16,000×g. Store sample at+2-8° C. prior to analysis.

NOTE: DP supernatant samples prepared in this way should be measuredneat in triplicate in the inactivated JEV ELISA.

e) Carefully remove as much of the residual supernatant from thecentrifuged tube using a 10-200 μL pipette without disturbing the alumpellet and discard the supernatant.

f) The pellet obtained is subjected to the Desorption procedure asdescribed below.

Drug Product Desorption Procedure

a) Add 158 μL of working strength desorption buffer to each pellet leftin the LoBind tube.

b) Resuspend the pellet by pipetting up and down several times to ensurecomplete re-suspension of the pellet and homogenisation of the sample.

c) Incubate samples for 10 min at RT on an orbital shaker at 500 rpm.

d) After incubation centrifuge samples at 3300×g for 5 minutes.

e) For each sample pipette 250 μL of 2×ELISA buffer into a fresh LoBindEppendorf tube.

f) Carefully remove 83.3 μL from the upper part of the supernatantcontaining the desorbed product without disturbing the pellet andtransfer into the tube containing the 2×ELISA buffer. Remove remainingsupernatant using a 20-200 μL pipette without disturbing the pellet anddiscard the supernatant.

g) Add another 158 μL of working strength desorption buffer to eachpellet.

h) Carry out 2 more desorption cycles (3 in total) pooling the 3×83.3 μLof the desorbed material+250 μL ELISA buffer into the appropriate tube.After the last step the remaining pellet can be discarded.

i) The final concentration of the viral antigen in the desorbed pool(s)should now be the same as the original 1 mL of DP from which it wasdesorbed.

Therefore, the concentration of inactivated JEV antigen content measuredin the desorbed pool can be directly related to the original DP.

Note: Analyse the desorbed samples by ELISA on the day of desorption.

j) Dilution of desorbed DP samples:

These will be tested at six dilutions in triplicate. An appropriatepre-dilution will be performed in the range of the standard curve e.g. 1in 15 (100 μL to 1400 μL diluent) or other suitable dilution, then sixdilutions of 1 in 15 pre-dilution will be made independently usingsample diluent.

2.7. Sample Loading and Plate Plan

Prepare samples and standards before analysis.

After blocking wash the plate using the plate washer employing the JEVELISA program. After that, remove any remaining wash buffer bydecanting. Invert the plate and blot it against a clean paper towel. Donot allow microtiter plate to dry between wash steps and reagentaddition.

Add 100 μL/well of standards/controls/samples and cover with cover plateand incubate for 1 hour+/−10 min at 37° C.

Add 100 μL of sample diluent to all wells not required for testing.

2.8. Preparation of Primary Antibody

Remove the required number of aliquots of the primary antibody from thefreezer and allow to thaw at RT. Prepare max 15 min before use Rabbitanti-JEV in sample diluent at a suitable dilution. Following sampleincubation, wash the plate using the plate washer employing the JEVELISA program. After that, remove any remaining wash buffer bydecanting. Invert the plate and blot it against a clean paper towel. Donot allow microtiter plate to dry between wash steps and reagentaddition.

Add 100 μL/well of diluted primary antibody, cover with cover plate andincubate for 1 hour+/−10 min at 37° C.

2.9. Preparation of Secondary Antibody Conjugate

Remove the required number of aliquots of the secondary antibodyconjugate from the freezer and allow to thaw at RT. Prepare max 15 minbefore use a dilution of Donkey anti-Rabbit-HRP in sample diluent; e.g.for a 1 in 10,000 dilution for make a 1 in 100 pre-dilution then make asecond dilution of 1 in 100.

Following primary antibody incubation, wash the plate using the platewasher employing the JEV ELISA program. After that, remove any remainingwash buffer by decanting. Invert the plate and blot it against a cleanpaper towel. Do not allow microtiter plate to dry between wash steps andreagent addition. Add 100 μL/well of diluted secondary antibodyconjugate, cover with cover plate and incubate for 1 hour+/−10 min at37° C.

2.10. Substrate Incubation

When the conjugate has been added, remove TMB from the 2-8° C.refrigerator. Pipette the required volume (12 mL of TMB per plate) intoa 50 mL centrifuge tube, using a serological pipette. Allow the TMB toreach room temperature in the dark. Following conjugate incubation washthe plate 3 times with the plate washer employing the JEV ELISA program.After that: remove any remaining wash buffer by decanting. Invert theplate and blot it against a clean paper towel. Do not allow microtiterplate to dry between wash steps and reagent addition. Add 100 μL/well ofTMB and develop the plate in the dark at Room Temperature for 10minutes.

2.11. Stopping and Reading

After 10 minutes of TMB incubation, stop the development by adding 100μL/well 2M sulphuric acid. Read the plate at 450 nm (reference filter630 nm) within 10 minutes of stopping using the BioTek reader and Gen5Secure software.

2.12. Data Analysis NIV/DS Data Analysis:

Gen5Secure software will be used to calculate the % Recovery of the QCs,concentration×dilutions, mean concentration of the samples corrected fordilutions and this value multiplied by 1.05 to correct for the additionof ELISA buffer.

DP Data Analysis:

Gen5Secure software will be used to calculate the % Recovery of the QCs,concentration×dilutions and mean concentration of the dilutions for thesamples.

For DP Supernatant Samples:

If the concentration of the supernatant sample is below the LLOQ of theassay (i.e. 0.05 AU/ml), then the supernatant sample should be recordedas <0.05 AU/ml

If the concentration of the supernatant sample is within the LOQs of theassay (i.e. 0.05 AU/ml to 1.25 AU/ml), the concentration value isrecorded for the supernatant sample.

If the concentration of the supernatant sample is above the ULOQ of theassay (i.e. 1.25 AU/ml), the preparation of the drug product supernatantshould be repeated. The supernatant sample will be re-tested byperforming a suitable pre-dilution into the range of the standard curvefollowed by 6 sample dilutions. (The desorbed Drug Product sample doesnot need to be repeated.) The mean concentration for the dilutions thatare within the LOQs of the assay (LLOQ 0.05 AU/ml to 1.25 AU/ml) will bethe recorded concentration value for the supernatant sample, providedthat the system suitability are met and at least 4 out of the 6 sampledilutions are within the LOQs.

2.13. Assay Acceptance Criteria

a) The correlation coefficient for the calibration curve must be >0.980.

b) % CVs≦15% for standards and samples (except DP supernatant) for thefour highest concentrations of the dilutions, % CV≦15% for controls

c) Individual blank ODs must be ≦0.2.

d) Assay controls must be within specified defined limits (for freshlyprepared controls 2 out of 3 QCs should have observed concentrationswithin ±30% of the nominal values; OR the levels set during QCqualification) for the assay to pass.

e) Assay validity will be recorded on the Gen5-print-out. If the platefails to meet the defined acceptance criteria the assay is deemedinvalid.

2.14. Reporting of Data NIV

a) Antigen content

The reported value for inactivated AU/mL is the mean of theconcentrations (which are within the LOQs of 0.04 to 1.25 AU/mL)calculated for the single sample dilutions corrected by the respectivedilution factors, and the mean multiplied by a correction factor of 1.05to account for the 5% volume of 20× ELISA buffer that was added to eachsample when it was taken. Antigen content will be recorded on Gen5print-out.

DS:

a) Identity

If the absorbances of the samples at lowest dilution (highestconcentration) are higher than 3 standard deviations above the meanvalue of the blank the result will be reported as positive

b) Antigen content

The reported value for inactivated AU/mL is the mean of theconcentrations (which are within the LOQs of 0.04 to 1.25 AU/mL)calculated for the single sample dilutions corrected by the respectivedilution factors, and the mean multiplied by a correction factor of 1.05to account for the 5% volume of 20× ELISA buffer that was added to eachsample when it was taken. Antigen content will be recorded on Gen5print-out.

Desorbed DP:

a) Identity:

If the absorbances of the samples at lowest dilution (highestconcentration) are higher than 3 standard deviations above the meanvalue of the blank the result will be reported as positive.

b) Antigen Content

The reported value for inactivated AU/mL is the mean of theconcentrations (which are within the LOQs of 0.05 to 0.8 AU/mL)calculated for the single sample dilutions corrected by the respectivedilution factors. Antigen content will be recorded on Gen5 print-out.

DP supernatant (degree of adsorption/degree of non-adsorption): Degreeof adsorption is reported in relationship to aluminium hydroxideformulated drug substance post filtration.

a) For calculation of the reported value, the reported antigen content(AU/mL) for the respective DS sample (post filtration) corrected for thedilution with aluminium hydroxide (5%) will be set at 100% and thepercentage of the concentration measured in the supernatant (correctedfor the addition of 5% ELISA buffer) calculated in relation to that. Thereportable value will be the difference between 100% and the percentagecalculated for the supernatant. Results will be reported to 2 decimalplaces.

${{Degree}\mspace{14mu} {of}\mspace{14mu} {adsorption}\mspace{11mu} (\%)} = {{100\%} - {\frac{{DP}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{AU}\text{/}{mL}} \right)*1.05}{{DS}\mspace{11mu} \left( {{AU}\text{/}{mL}} \right)*0.95}*100\%}}$

The degree of non-adsorption will be calculated as detailed below andresults will be reported to 2 decimal places:

${{Degree}\mspace{14mu} {of}\mspace{14mu} {non}\text{-}{adsorption}\mspace{11mu} (\%)} = {\frac{{DP}\mspace{14mu} {supernatant}\mspace{14mu} \left( {{AU}\text{/}{mL}} \right)*1.05}{{DS}\mspace{11mu} \left( {{AU}\text{/}{mL}} \right)*0.95}*100\%}$

b) In case the neat supernatant does not contain any measurable antigen(ie. observed supernatant concentration less than LLOQ, where LLOQ=0.05AU/mL), the LLOQ will be used for the calculation of the result. Theresult in this case is reported as “greater than x %”. For example ifthe DS sample is measured as 12.00 AU/mL and no signal was measured inthe supernatant; with the LLOQ of 0.05 AU/mL then amount in supernatantis <0.05*1.05=<0.0525 AU/mL. The amount of DS after buffer correction is12.00*0.95=11.40 AU/mL, and the reported result for degree of adsorptionis <100-0.0525/11.4*100=>99.54%. The degree of non-adsorption will alsobe reported (i.e. 100—the % degree of adsorption).

Example 5 Introduction

In order to further investigate the mode of action that leads to productinstability/potency loss of the JEV vaccine,Ala-(His)6-OprF190-342-OprI21-83 (SEQ ID NO: 1, FIG. 14)—herein alsoreferred to as “protein A” was used in a preliminary screening assayincorporating ALHYDROGEL® lots with different metal content and spikingwith copper ions and sulfite.

Material:

-   -   Copper(II)chloride dihydrate (Sigma, Order no. 807483)    -   10×PBS (Gibco, Order No. 14200-091)    -   15 ml Falcon tubes (Greiner, Cat. No. 188724)    -   Incubator Infors HT Incubator Multitron Standard (InforsAG)    -   Aqua bidest. (Fresenius Kabi, Art no. 0712221/01 A)

Preparation of Stock Solutions:

-   -   Copper(II) stock solution        -   20 mM Copper(II) stock solution was prepared by dissolving            341 mg of Copper(II)chloride dihydrate in 100 mL of aqua            bidest.    -   Sodiummetabisulfite stock solution        -   200 mM Sodiummetabisulfite stock solution was prepared by            dissolving 1.52 g of Sodiummetabisulfite in 35 mL PBS. This            solution was adjusted the pH to 7.3 with NaOH and filled up            to a volume of 40 mL with PBS. The solution was them            filtered via 0.2μ syringe filter.

Preparation of Working Solutions

-   -   Working solutions were prepared by dilution of metal stock        solutions with aqua bidest. and sterile filtration via 0.2μ        syringe filter. (Mini Kleenpak 25 mm-Pall)

Preparation of Buffer Solutions

-   -   ⅓ PBS+0.9% NaCl        -   1×PBS buffer solution was prepared by 1:10 dilution of            10×PBS with aqua bidest. The pH of this buffer solution was            7.5. PBS buffer solutions adjusted to pH 7.3 and 8.0 were            prepared by adjusting the pH with HCl or NaOH respectively.        -   9 g of NaCl were dissolved in 333 mL of either pH 7.3 of pH            8.0 buffer solution and then brought to 1000 mL with aqua            bidest. followed by filtration via 0.2μ bottletop filter.

Sample Preparation:

Formulations of Protein A and different lots of ALHYDROGEL® (Lot 4230 &Lot 4074) were prepared in ⅓ PBS+0.9% NaCl at two different pH valuesand were spiked with sulfite according to the following scheme:

Sample Spike Alum Cu(II) Sulfit No. Name pH batch [ng/mL] [mM] 117112011_PROTEIN 7.3 4074 A_4074_ref_4°_pH 7.3 2 17112011_PROTEIN 7.34074 A_4074_ref_37°_pH 7.3 3 17112011_PROTEIN 7.3 4074 1A_4074_Sulfit_37°_pH 7.3 4 17112011_PROTEIN 7.3 4230 A_4230_ref_4°_pH7.3 5 17112011_PROTEIN 7.3 4230 A_4230_ref_37°_pH 7.3 6 17112011_PROTEIN7.3 4230 1 A_4230_Sulfit_37°_pH 7.3 7 17112011_PROTEIN 8 4074A_4074_ref_4°_pH 8 8 17112011_PROTEIN 8 4074 A_4074_ref_37°_pH 8 917112011_PROTEIN 8 4074 1 A_4074_Sulfit_37°_pH 8 10 17112011_PROTEIN 84230 A_4230_ref_4°_pH 8 11 17112011_PROTEIN 8 4230 A_4230_ref_37°_pH 812 17112011_PROTEIN 8 4230 1 A_4230_Sulfit_37°_pH 8

Samples 1, 6, 11 and 16 were stored at 4° C. (reference samples). Allother samples were incubated at 37° C. for 96 hours.

After 96 hours all samples were subjected to a desorption procedure toseparate the antigen from ALHYDROGEL®. The desorbed antigen was analyzedby RPC.

Results:

Results showed severe degradation of the antigen Protein A in thepresence of sulfite. The degradation was more pronounced in the samplesformulated with ALHYDROGEL® of higher metal impurity content.

TABLE 1 Metal ion content in Aluminium hydroxide lot 4230 and 4074analyzed by ICP-MS. Alum Lot Al Cr Fe Co Ni Cu Ag Cd W Pb V Rb Mo (2%solution) μg/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mLng/mL ng/mL ng/mL Alum Lot 4230 9570 1139 5640 7 816 64 <5 <5 <25 24 13<5 11 RQCS 0890 Alum Lot 4074 9130 20 266 <5 15 <25 <5 <5 <25 19 <5 <5<5 RQCS0013 Mix 50/50% of 9130 579 2952 <5.8 415 <45 <5 <5 <25 21.6 <9<5 <8 both Lots* *Calculated residual metal content

TABLE 2 Pipetting scheme of DOE Sample % % μg/mL U ppm No. Name pH NIVdil Alum 4230 Alum 4074 PS Frag. Stock Extractables Stock CH2O  120110819_DOE_spl_1 7 2 100 0 50 4.2 0  2 20110819_DOE_spl_2 7 2 0 100 04.2 0  3 20110819_DOE_spl_3 7 2 0 100 0 0 40  4 20110819_DOE_spl_4 8 2 0100 50 4.2 0  5 20110819_DOE_spl_5 7.5 2 50 50 50 0 0  620110819_DOE_spl_6 7 2 0 100 50 0 40  7 20110819_DOE_spl_7 7 2 100 0 500 0  8 20110819_DOE_spl_8 8 2 0 100 50 0 0  9 20110819_DOE_spl_9 8 2 1000 0 0 0 10 20110819_DOE_spl_10 8 2 100 0 50 0 0 11 20110819_DOE_spl_11 82 0 100 50 4.2 40 12 20110819_DOE_spl_12 7 2 100 0 0 4.2 0 1320110819_DOE_spl_13 8 2 0 100 0 0 40 14 20110819_DOE_spl_14 7 2 100 0 00 40 15 20110819_DOE_spl_15 8 2 0 100 0 4.2 0 16 20110819_DOE_spl_16 8 2100 0 50 0 40 17 20110819_DOE_spl_17 7 2 0 100 50 4.2 0 1820110819_DOE_spl_18 7 2 0 100 50 4.2 40 19 20110819_DOE_spl_19 8 2 100 00 4.2 40 20 20110819_DOE_spl_20 8 2 0 100 0 4.2 40 2120110819_DOE_spl_21 8 2 0 100 50 0 40 22 20110819_DOE_spl_22 8 2 100 0 04.2 0 23 20110819_DOE_spl_23 7 2 0 100 0 4.2 40 24 20110819_DOE_spl_24 72 100 0 50 0 40 25 20110819_DOE_spl_25 7 2 0 100 50 0 0 2620110819_DOE_spl_26 7 2 0 100 0 0 0 27 20110819_DOE_spl_27 8 2 100 0 504.2 0 28 20110819_DOE_spl_28 8 2 100 0 0 0 40 29 20110819_DOE_spl_29 8 2100 0 50 4.2 40 30 20110819_DOE_spl_30 7 2 100 0 0 0 0 3120110819_DOE_spl_31 7.5 2 50 50 0 0 0 32 20110819_DOE_spl_32 8 2 0 100 00 0 33 20110819_DOE_spl_33 7 2 100 0 50 4.2 40 34 20110819_DOE_spl_34 72 100 0 0 4.2 40 Sample Volume [μl] No. Name pH NIV Buffer Alum 4230Alum 4074 PS Frag. Stock Extractables Stock CH2O Total  120110819_DOE_spl_1 7 2500 1812 250 0 125 313 0 5000  220110819_DOE_spl_2 7 2500 1937 0 250 0 313 0 5000  3 20110819_DOE_spl_37 2500 2228 0 250 0 0 22 5000  4 20110819_DOE_spl_4 8 2500 1812 0 250125 313 0 5000  5 20110819_DOE_spl_5 7.5 2500 2125 125 125 125 0 0 5000 6 20110819_DOE_spl_6 7 2500 2103 0 250 125 0 22 5000  720110819_DOE_spl_7 7 2500 2125 250 0 125 0 0 5000  8 20110819_DOE_spl_88 2500 2125 0 250 125 0 0 5000  9 20110819_DOE_spl_9 8 2500 2250 250 0 00 0 5000 10 20110819_DOE_spl_10 8 2500 2125 250 0 125 0 0 5000 1120110819_DOE_spl_11 8 2500 1790 0 250 125 313 22 5000 1220110819_DOE_spl_12 7 2500 1937 250 0 0 313 0 5000 1320110819_DOE_spl_13 8 2500 2228 0 250 0 0 22 5000 14 20110819_DOE_spl_147 2500 2228 250 0 0 0 22 5000 15 20110819_DOE_spl_15 8 2500 1937 0 250 0313 0 5000 16 20110819_DOE_spl_16 8 2500 2103 250 0 125 0 22 5000 1720110819_DOE_spl_17 7 2500 1812 0 250 125 313 0 5000 1820110819_DOE_spl_18 7 2500 1790 0 250 125 313 22 5000 1920110819_DOE_spl_19 8 2500 1915 250 0 0 313 22 5000 2020110819_DOE_spl_20 8 2500 1915 0 250 0 313 22 5000 2120110819_DOE_spl_21 8 2500 2103 0 250 125 0 22 5000 2220110819_DOE_spl_22 8 2500 1937 250 0 0 313 0 5000 2320110819_DOE_spl_23 7 2500 1915 0 250 0 313 22 5000 2420110819_DOE_spl_24 7 2500 2103 250 0 125 0 22 5000 2520110819_DOE_spl_25 7 2500 2125 0 250 125 0 0 5000 2620110819_DOE_spl_26 7 2500 2250 0 250 0 0 0 5000 27 20110819_DOE_spl_278 2500 1812 250 0 125 313 0 5000 28 20110819_DOE_spl_28 8 2500 2228 2500 0 0 22 5000 29 20110819_DOE_spl_29 8 2500 1790 250 0 125 313 22 500030 20110819_DOE_spl_30 7 2500 2250 250 0 0 0 0 5000 3120110819_DOE_spl_31 7.5 2500 2250 125 125 0 0 0 5000 3220110819_DOE_spl_32 8 2500 2250 0 250 0 0 0 5000 33 20110819_DOE_spl_337 2500 1790 250 0 125 313 22 5000 34 20110819_DOE_spl_34 7 2500 1915 2500 0 313 22 5000 sum: 85000 69014 4250 4250 2125 5015 346 170000

TABLE 3 Comparison of leachables from stopper extract and JEV09L37 SN.All peaks with an area of >0.1 mAU · min are included in this table.Stopper extract Stopper concentrate FVL Relative Retention extract 1:16diluted in L37 concentration time concentrate formulation SN compared toFVL (min) Peak area (mAU · min) (%) 12.40 0.79 0.05 0.10 47 13.14 1.910.12 n.d. additional peak compared to FVL 13.48 0.81 0.05 n.d.additional peak compared to FVL 13.74 0.43 0.03 n.d. additional peakcompared to FVL 14.10 0.75 0.05 n.d. additional peak compared to FVL14.41 0.49 0.03 0.25 12 16.12 29.64 1.85 0.45 414  16.71 2.75 0.17 n.d.additional peak compared to FVL 17.15 14.68 0.92 0.11 815  18.68 0.900.06 0.57 10 20.08 0.70 0.04 0.25 18 20.75 0.42 0.03 n.d. additionalpeak compared to FVL 21.27 0.54 0.03 n.d. additional peak compared toFVL 22.13 2.84 0.18 0.15 122  22.74 0.55 0.03 0.17 20 23.74 1.74 0.110.27 41 24.88 0.98 0.06 0.12 51 27.63 0.84 0.05 0.16 32 29.46 0.72 0.04n.d. additional peak compared to FVL 31.40 0.39 0.02 n.d. additionalpeak compared to FVL 35.23 4.70 0.29 0.41 72 SUM 67.59 4.2 3.0  140 

TABLE 4 DOE results obtained after 4 and 8 weeks at 22° C. Antigen wasdesorbed from Alum and analysed by ELISA (monoclonal and polyclonal).*(x-fold increase compared to FVL) 4 weeks at 22° C. 8 weeks at 22° C.Spiked PS Spiked Mono. Poly. Mono. Poly. Alum 4230 Frag. Spiked FormalinELISA ELISA ELISA ELISA Sample pH (%) (μg/mL) leachables* (ppm) (AU/mL)(AU/mL) Ratio (AU/mL) (AU/mL) Ratio 1 7 100 50 1.4 0 9.725 12.814 0.766.342 3.649 0.575 2 7 0 0 1.4 0 15.254 16.331 0.93 9.865 8.039 0.815 3 70 0 0 40 12.457 12.614 0.99 9.037 8.176 0.905 4 8 0 50 1.4 0 13.51312.971 1.04 10.328 9.533 0.923 5 7.5 50 50 0 0 13.592 14.924 0.91 10.3588.032 0.775 6 7 0 50 0 40 12.942 13.878 0.93 10.008 9.896 0.989 7 7 10050 0 0 9.649 12.608 0.77 6.089 4.092 0.672 8 8 0 50 0 0 11.184 11.9020.94 9.113 9.048 0.993 9 8 100 0 0 0 11.436 12.883 0.89 8.858 7.4090.836 10 8 100 50 0 0 13.361 15.516 0.86 10.525 8.158 0.775 11 8 0 501.4 40 13.209 13.608 0.97 9.349 9.201 0.984 12 7 100 0 1.4 0 8.4 11.9130.71 5.001 2.951 0.590 13 8 0 0 0 40 10.294 10.483 0.98 8.019 8.2841.033 14 7 100 0 0 40 9.972 12.015 0.83 7.435 5.802 0.780 15 8 0 0 1.4 014.096 15.618 0.9 10.192 9.531 0.935 16 8 100 50 0 40 9.78 13.514 0.7211.011 8.947 0.813 17 7 0 50 1.4 0 11.213 14.285 0.78 10.246 8.377 0.81818 7 0 50 1.4 40 11.183 11.499 0.97 10.539 9.217 0.875 19 8 100 0 1.4 4010.536 10.935 0.96 10.151 8.341 0.822 20 8 0 0 1.4 40 10.026 9.654 1.0411.306 9.281 0.821 21 8 0 50 0 40 10.149 9.986 1.02 11.213 9.205 0.82122 8 100 0 1.4 0 10.051 12.193 0.82 10.918 6.841 0.627 23 7 0 0 1.4 4010.024 10.74 0.93 10.711 7.963 0.743 24 7 100 50 0 40 9.535 10.254 0.9310.642 6.991 0.657 25 7 0 50 0 0 11.143 11.765 0.95 13.054 8.684 0.66526 7 0 0 0 0 11.431 11.796 0.97 13.753 10.391 0.756 27 8 100 50 1.4 010.09 11.953 0.84 11.506 7.334 0.637 28 8 100 0 0 40 10.137 11.223 0.911 8.131 0.739 29 8 100 50 1.4 40 9.605 10.535 0.91 10.848 7.939 0.73230 7 100 0 0 0 6.485 8.379 0.77 7.957 3.523 0.443 31 7.5 50 0 0 0 11.08112.229 0.91 12.377 9.535 0.770 32 8 0 0 0 0 11.421 12.002 0.95 11.369.859 0.868 33 7 100 50 1.4 40 9.03 10.583 0.85 9.753 6.571 0.674 34 7100 0 1.4 40 9.05 10.927 0.83 11.715 6.872 0.587

TABLE 5 Analysis of Variance for ratio Monoclonal/Polyclonal ELISA afterstorage 4 weeks at 22° C. Analysis of Variance for Ratio 4 weeks Sum ofMean F- P- Source Squares Df Square Ratio Value A: pH 0.02205 1 0.022055.48 0.0326 B: Alum 0.117612 1 0.117612 29.20 0.0001 C: PS Fragments0.0008 1 0.0008 0.20 0.6618 D: Extractables 0.0008 1 0.0008 0.20 0.6618E: Formaline 0.0242 1 0.0242 6.01 0.0261 AB 0.0001125 1 0.0001125 0.030.8694 AC 0.00045 1 0.00045 0.11 0.7425 AD 0.01125 1 0.01125 2.79 0.1141AE 0.00405 1 0.00405 1.01 0.3309 BC 0.0000125 1 0.0000125 0.00 0.9563 BD0.0010125 1 0.0010125 0.25 0.6229 BE 0.0006125 1 0.0006125 0.15 0.7017CD 0.0008 1 0.0008 0.20 0.6618 CE 0.0008 1 0.0008 0.20 0.6618 DE 0.00721 0.0072 1.79 0.1999 Total error 0.0644375 16 0.00402734 Total (corr.)0.2562 31 R-squared = 74.8488 percent R-squared (adjusted for d.f.) =51.2695 percent Standard Error of Est. = 0.0634614 Mean absolute error =0.0352734 Durbin-Watson statistic = 1.47556

TABLE 6 Analysis of Variance for ratio Monoclonal/Polyclonal ELISA afterstorage at 22° C. for 8 weeks. Analysis of Variance for Ratio 8 weeksSum of Mean F- P- Source Squares Df Square Ratio Value A: pH 0.102378 10.102378 11.19 0.0041 B: Alum 0.275653 1 0.275653 30.13 0.0000 C: PSFragments 0.00300312 1 0.00300312 0.33 0.5747 D: Extractables 0.01087811 0.0108781 1.19 0.2917 E: Formaline 0.0318781 1 0.0318781 3.48 0.0804AB 0.00137813 1 0.00137813 0.15 0.7031 AC 0.00382812 1 0.00382812 0.420.5269 AD 0.00137813 1 0.00137813 0.15 0.7031 AE 0.0166531 1 0.01665311.82 0.1961 BC 0.000253125 1 0.000253125 0.03 0.8700 BD 0.00382813 10.00382813 0.42 0.5269 BE 0.00195313 1 0.00195313 0.21 0.6503 CD0.00195313 1 0.00195313 0.21 0.6503 CE 0.000153125 1 0.000153125 0.020.8987 DE 0.00525313 1 0.00525313 0.57 0.4596 Total error 0.1464 160.00915 Total (corr.) 0.606822 31 R-squared = 75.8743 percent R-squared(adjusted for d.f.) = 53.2565 percent Standard Error of Est. = 0.0956556Mean absolute error = 0.0571484 Durbin-Watson statistic = 0.888586

TABLE 7 Regression analysis for “Ratio 8 weeks” including pH, Alum andFormaldehyde. Regression coeffs. for Ratio 8 weeks constant = 0.0228125A: pH = 0.113125 B: Alum = −0.00185625 E: Formaline = 0.0315625

TABLE 8 Estimation of results “Ratio 8 weeks” generated using the fittedmodel. Estimation Results for Ratio 8 weeks Observed Fitted Lower 95.0%Upper 95.0% Row Value Value CL for Mean CL for Mean 1 0.58 0.59750.536766 0.658234 2 0.81 0.783125 0.722391 0.843859 3 0.9 0.846250.785516 0.906984 4 0.92 0.89625 0.835516 0.956984 6 0.99 0.846250.785516 0.906984 7 0.67 0.5975 0.536766 0.658234 8 0.99 0.896250.835516 0.956984 9 0.84 0.710625 0.649891 0.771359 10 0.78 0.7106250.649891 0.771359 11 0.98 0.959375 0.898641 1.02011 12 0.59 0.59750.536766 0.658234 13 1.03 0.959375 0.898641 1.02011 14 0.78 0.6606250.599891 0.721359 15 0.94 0.89625 0.835516 0.956984 16 0.81 0.773750.713016 0.834484 17 0.82 0.783125 0.722391 0.843859 18 0.88 0.846250.785516 0.906984 19 0.82 0.77375 0.713016 0.834484 20 0.82 0.9593750.898641 1.02011 21 0.82 0.959375 0.898641 1.02011 22 0.63 0.7106250.649891 0.771359 23 0.74 0.84625 0.785516 0.906984 24 0.66 0.6606250.599891 0.721359 25 0.67 0.783125 0.722391 0.843859 26 0.76 0.7831250.722391 0.843859 27 0.64 0.710625 0.649891 0.771359 28 0.74 0.773750.713016 0.834484 29 0.73 0.77375 0.713016 0.834484 30 0.44 0.59750.536766 0.658234 32 0.87 0.89625 0.835516 0.956984 33 0.67 0.6606250.599891 0.721359 34 0.59 0.660625 0.599891 0.721359

TABLE 9 Plan for experiment 20110913(NIV) Pipetting plan Stock Sol [mM]Total Volume: 2000 μl Ni(II)SO4 1 NIV material: 11A74 Cu(II)Cl2 1Cr(III)Cl 1 μg/L Sample fragmented No. Name pH Ni(II)SO4 Cu(II)Cl2Cr(III) PS  1 NIV_unspiked_pH7_22° C. 7  2 NIV_Ni(II)_100_pH7_22° C. 7100  3 NIV_Ni(II)_500_pH7_22° C. 7 500  4 NIV_Ni(II)_1000_pH7_22° C. 71000  5 NIV_Cu(II)_100_pH7_22° C. 7 100  6 NIV_Cu(II)_500_pH7_22° C. 7500  7 NIV_Cu(II)_1000_pH7_22° C. 7 1000  8 NIV_CR(III)_100_pH7_22° C. 7100  9 NIV_CR(III)_500_pH7_22° C. 7 500 10 NIV_CR(III)_1000_pH7_22° C. 71000 11 NIV_PS spike_pH7_22° C. 7 50 12 NIV_Ni(II)_100_PSspike_pH7_22°C. 7 100 50 13 NIV_Ni(II)_500_PSspike_pH7_22° C. 7 500 50 14NIV_Ni(II)_1000_PSspike_pH7_22° C. 7 1000 50 15NIV_Cu(II)_100_PSspike_pH7_22° C. 7 100 50 16NIV_Cu(II)_500_PSspike_pH7_22° C. 7 500 50 17NIV_Cu(II)_1000_PSspike_pH7_22° C. 7 1000 50 18NIV_CR(III)_100_PSspike_pH7_22° C. 7 100 50 19NIV_CR(III)_500_PSspike_pH7_22° C. 7 500 50 20NIV_CR(III)_1000_PSspike_pH7_22° C. 7 1000 50 21 NIV_unspiked_pH8_22° C.8 22 NIV_Ni(II)_100_pH8_22° C. 8 100 23 NIV_Ni(II)_500_pH8_22° C. 8 50024 NIV_Ni(II)_1000_pH8_22° C. 8 1000 25 NIV_Cu(II)_100_pH8_22° C. 8 10026 NIV_Cu(II)_500_pH8_22° C. 8 500 27 NIV_Cu(II)_1000_pH8_22° C. 8 100028 NIV_CR(III)_100_pH8_22° C. 8 100 29 NIV_CR(III)_500_pH8_22° C. 8 50030 NIV_CR(III)_1000_pH8_22° C. 8 1000 31 NIV_PS spike_pH8_22° C. 8 50 32NIV_Ni(II)_100_PSspike_pH8_22° C. 8 100 50 33NIV_Ni(II)_500_PSspike_pH8_22° C. 8 500 50 34NIV_Ni(II)_1000_PSspike_pH8_22° C. 8 1000 50 35NIV_Cu(II)_100_PSspike_pH8_22° C. 8 100 50 36NIV_Cu(II)_500_PSspike_pH8_22° C. 8 500 50 37NIV_Cu(II)_1000_PSspike_pH8_22° C. 8 1000 50 38NIV_CR(III)_100_PSspike_pH8_22° C. 8 100 50 39NIV_CR(III)_500_PSspike_pH8_22° C. 8 500 50 40NIV_CR(III)_1000_PSspike_pH8_22° C. 8 1000 50 MW [g/Mol] otheradditives: Ni 58.7 Stock Sol: Cu 63.6 frag PS 2 mg/mL Cr 52.0 Volume[μl] Sample fragmented No. Name pH NIV PBS Ni(II)SO4 Cu(II)Cl2 Cr(III)PS Total  1 NIV_unspiked_pH7_22° C. 7 1800 200 0 0 0 0 2000  2NIV_Ni(II)_100_pH7_22° C. 7 1800 197 3 0 0 0 2000  3NIV_Ni(II)_500_pH7_22° C. 7 1800 183 17 0 0 0 2000  4NIV_Ni(II)_1000_pH7_22° C. 7 1800 166 34 0 0 0 2000  5NIV_Cu(II)_100_pH7_22° C. 7 1800 197 0 3 0 0 2000  6NIV_Cu(II)_500_pH7_22° C. 7 1800 184 0 16 0 0 2000  7NIV_Cu(II)_1000_pH7_22° C. 7 1800 169 0 31 0 0 2000  8NIV_CR(III)_100_pH7_22° C. 7 1800 196 0 0 4 0 2000  9NIV_CR(III)_500_pH7_22° C. 7 1800 181 0 0 19 0 2000 10NIV_CR(III)_1000_pH7_22° C. 7 1800 162 0 0 38 0 2000 11 NIV_PSspike_pH7_22° C. 7 1800 150 0 0 0 50 2000 12NIV_Ni(II)_100_PSspike_pH7_22° C. 7 1800 147 3 0 0 50 2000 13NIV_Ni(II)_500_PSspike_pH7_22° C. 7 1800 133 17 0 0 50 2000 14NIV_Ni(II)_1000_PSspike_pH7_22° C. 7 1800 116 34 0 0 50 2000 15NIV_Cu(II)_100_PSspike_pH7_22° C. 7 1800 147 0 3 0 50 2000 16NIV_Cu(II)_500_PSspike_pH7_22° C. 7 1800 134 0 16 0 50 2000 17NIV_Cu(II)_1000_PSspike_pH7_22° C. 7 1800 119 0 31 0 50 2000 18NIV_CR(III)_100_PSspike_pH7_22° C. 7 1800 146 0 0 4 50 2000 19NIV_CR(III)_500_PSspike_pH7_22° C. 7 1800 131 0 0 19 50 2000 20NIV_CR(III)_1000_PSspike_pH7_22° C. 7 1800 112 0 0 38 50 2000 21NIV_unspiked_pH8_22° C. 8 1800 200 0 0 0 0 2000 22NIV_Ni(II)_100_pH8_22° C. 8 1800 197 3 0 0 0 2000 23NIV_Ni(II)_500_pH8_22° C. 8 1800 183 17 0 0 0 2000 24NIV_Ni(II)_1000_pH8_22° C. 8 1800 166 34 0 0 0 2000 25NIV_Cu(II)_100_pH8_22° C. 8 1800 197 0 3 0 0 2000 26NIV_Cu(II)_500_pH8_22° C. 8 1800 184 0 16 0 0 2000 27NIV_Cu(II)_1000_pH8_22° C. 8 1800 169 0 31 0 0 2000 28NIV_CR(III)_100_pH8_22° C. 8 1800 196 0 0 4 0 2000 29NIV_CR(III)_500_pH8_22° C. 8 1800 181 0 0 19 0 2000 30NIV_CR(III)_1000_pH8_22° C. 8 1800 162 0 0 38 0 2000 31 NIV_PSspike_pH8_22° C. 8 1800 150 0 0 0 50 2000 32NIV_Ni(II)_100_PSspike_pH8_22° C. 8 1800 147 3 0 0 50 2000 33NIV_Ni(II)_500_PSspike_pH8_22° C. 8 1800 133 17 0 0 50 2000 34NIV_Ni(II)_1000_PSspike_pH8_22° C. 8 1800 116 34 0 0 50 2000 35NIV_Cu(II)_100_PSspike_pH8_22° C. 8 1800 147 0 3 0 50 2000 36NIV_Cu(II)_500_PSspike_pH8_22° C. 8 1800 134 0 16 0 50 2000 37NIV_Cu(II)_1000_PSspike_pH8_22° C. 8 1800 119 0 31 0 50 2000 38NIV_CR(III)_100_PSspike_pH8_22° C. 8 1800 146 0 0 4 50 2000 39NIV_CR(III)_500_PSspike_pH8_22° C. 8 1800 131 0 0 19 50 2000 40NIV_CR(III)_1000_PSspike_pH8_22° C. 8 1800 112 0 0 38 50 2000

TABLE 10 Plan for experiment 20110913(DP) Pipetting plan Stock Sol [mM]Total Volume: 10000 μl Ni(II)SO4 1 Cu(II)Cl2 1 DP: 11D87 bulk Cr(III)Cl31 Sample μg/L No. Name pH Ni(II)SO4 Cu(II)Cl2 Cr(III) fragmented PS 1DP_unspiked_pH7 7 2 DP_Ni(II)_100_pH7 7 100 3 DP_Ni(II)_500_pH7 7 500 4DP_Ni(II)_1000_pH7 7 1000 5 DP_Cu(II)_100_pH7 7 100 6 DP_Cu(II)_500_pH77 500 7 DP_Cu(II)_1000_pH7 7 1000 8 DP_Cr(III)_100_pH7 7 100 9DP_Cr(III)_500_pH7 7 500 10 DP_Cr(III)_1000_pH7 7 1000 11DP_unspiked_pH8 8 12 DP_Ni(II)_100_pH8 8 100 13 DP_Ni(II)_500_pH8 8 50014 DP_Ni(II)_1000_pH8 8 1000 15 DP_Cu(II)_100_pH8 8 100 16DP_Cu(II)_500_pH8 8 500 17 DP_Cu(II)_1000_pH8 8 1000 18DP_Cr(III)_100_pH8 8 100 19 DP_Cr(III)_500_pH8 8 500 20DP_Cr(III)_1000_pH8 8 1000 MW [g/Mol] other additives: Ni 58.7 StockSol: Cu 63.6 frag PS 2 mg/mL Cr 52.0 Sample Volume [μl] No. Name pH DPBuffer Ni(II)SO4 Cu(II)Cl2 Cr(III) fragmented PS Total 1 DP_unspiked_pH77 9500 500 0 0 0 0 10000 2 DP_Ni(II)_100_pH7 7 9500 483 17 0 0 0 10000 3DP_Ni(II)_500_pH7 7 9500 415 85 0 0 0 10000 4 DP_Ni(II)_1000_pH7 7 9500330 170 0 0 0 10000 5 DP_Cu(II)_100_pH7 7 9500 484 0 16 0 0 10000 6DP_Cu(II)_500_pH7 7 9500 421 0 79 0 0 10000 7 DP_Cu(II)_1000_pH7 7 9500343 0 157 0 0 10000 8 DP_Cr(III)_100_pH7 7 9500 481 0 0 19 0 10000 9DP_Cr(III)_500_pH7 7 9500 404 0 0 96 0 10000 10 DP_Cr(III)_1000_pH7 79500 308 0 0 192 0 10000 11 DP_unspiked_pH8 8 9500 500 0 0 0 0 10000 12DP_Ni(II)_100_pH8 8 9500 483 17 0 0 0 10000 13 DP_Ni(II)_500_pH8 8 9500415 85 0 0 0 10000 14 DP_Ni(II)_1000_pH8 8 9500 330 170 0 0 0 10000 15DP_Cu(II)_100_pH8 8 9500 484 0 16 0 0 10000 16 DP_Cu(II)_500_pH8 8 9500421 0 79 0 0 10000 17 DP_Cu(II)_1000_pH8 8 9500 343 0 157 0 0 10000 18DP_Cr(III)_100_pH8 8 9500 481 0 0 19 0 10000 19 DP_Cr(III)_500_pH8 89500 404 0 0 96 0 10000 20 DP_Cr(III)_1000_pH8 8 9500 308 0 0 192 010000

TABLE 11 Plan for experiment 20110812-Metal Ion Spiked DP Pipetting planStock Sol [mM] MW [g/Mol] FVL ng/mL μM Total Volume: 30000 μl Fe(II)Cl31 Fe 55.9 282 5.049 DP: 11D87 bulk Fe(III)Cl3 1 Fe 55.9 282 5.045Ni(II)SO4 1 Ni 58.7 41 0.698 Co(II)Cl2 1 Co 58.9 0.33 0.006 Cu(II)Cl2 1Cu 63.6 3 0.047 Zn(II)SO4 1 Zn 65.4 Cr(III)Cl3 1 Cr 52.0 Sample μg/L No.Name pH DP dil Fe(II)Cl3 Fe(III)Cl3 Ni(II)SO4 Co(II)Cl2 Cu(II)Cl2Zn(II)SO4 1 DP_Fe(II)_pH7 7 1 500 2 DP_Fe(III)_pH7 7 1 500 3DP_Ni(II)_pH7 7 1 500 4 DP_Co(II)_pH7 7 1 500 5 DP_Cu(II)_pH7 7 1 500 6DP_Zn_pH7 7 1 500 7 DP_metalmix_pH7 7 1 500 500 500 500 500 500 8DP_unspiked_pH7 7 1 9 DP_Fe(II)_pH7.4 7.4 1 500 10 DP_Fe(III)_pH7.4 7.41 500 11 DP_Ni(II)_pH7.4 7.4 1 500 12 DP_Co(II)_pH7.4 7.4 1 500 13DP_Cu(II)_pH7.4 7.4 1 500 14 DP_Zn_pH7.4 7.4 1 500 15 DP_metalmix_pH7.47.4 1 500 500 500 500 500 500 16 DP_unspiked_pH7.4 7.4 1 17DP_Fe(II)_pH7.8 7.8 1 500 18 DP_Fe(III)_pH7.8 7.8 1 500 19DP_Ni(II)_pH7.8 7.8 1 500 20 DP_Co(II)_pH7.8 7.8 1 500 21DP_Cu(II)_pH7.8 7.8 1 500 22 DP_Zn_pH7.8 7.8 1 500 23 DP_metalmix_pH7.87.8 1 500 500 500 500 500 500 24 DP_unspiked_pH7.8 7.8 1 Sample μg/L No.Name pH DP dil Cr(III) 25 DP_CR(III)_pH7 7 1 500 26 DP_Cr(III)_pH7.4 7.41 500 27 DP_Cr(III)_pH7.8 7.8 1 500 Sample Volume [μl] No. Name pH NIVdil Buffer Fe(II)Cl3 Fe(III)Cl3 Ni(II)SO4 Co(II)Cl2 Cu(II)Cl2 Zn(II)SO4Total 1 DP_Fe(II)_pH7 7 27750 1981 269 0 0 0 0 0 30000 2 DP_Fe(III)_pH77 27750 1982 0 268 0 0 0 0 30000 3 DP_Ni(II)_pH7 7 27750 1981 0 0 269 00 0 30000 4 DP_Co(II)_pH7 7 27750 1994 0 0 0 256 0 0 30000 5DP_Cu(II)_pH7 7 27750 1995 0 0 0 0 255 0 30000 6 DP_Zn_pH7 7 27750 20140 0 0 0 0 236 30000 7 DP_metalmix_pH7 7 27750 698 269 268 269 256 255236 30000 8 DP_unspiked_pH7 7 27750 2250 0 0 0 0 0 0 30000 9DP_Fe(II)_pH7.4 7.4 27750 1981 269 0 0 0 0 0 30000 10 DP_Fe(III)_pH7.47.4 27750 1982 0 268 0 0 0 0 30000 11 DP_Ni(II)_pH7.4 7.4 27750 1981 0 0269 0 0 0 30000 12 DP_Co(II)_pH7.4 7.4 27750 1994 0 0 0 256 0 0 30000 13DP_Cu(II)_pH7.4 7.4 27750 1995 0 0 0 0 255 0 30000 14 DP_Zn_pH7.4 7.427750 2014 0 0 0 0 0 236 30000 15 DP_metalmix_pH7.4 7.4 27750 698 269268 269 256 255 236 30000 16 DP_unspiked_pH7.4 7.4 27750 2250 0 0 0 0 00 30000 17 DP_Fe(II)_pH7.8 7.8 27750 1981 269 0 0 0 0 0 30000 18DP_Fe(III)_pH7.8 7.8 27750 1982 0 268 0 0 0 0 30000 19 DP_Ni(II)_pH7.87.8 27750 1981 0 0 269 0 0 0 30000 20 DP_Co(II)_pH7.8 7.8 27750 1994 0 00 256 0 0 30000 21 DP_Cu(II)_pH7.8 7.8 27750 1995 0 0 0 0 255 0 30000 22DP_Zn_pH7.8 7.8 27750 2014 0 0 0 0 0 236 30000 23 DP_metalmix_pH7.8 7.827750 698 269 268 269 256 255 236 30000 24 DP_unspiked_pH7.8 7.8 277502250 0 0 0 0 0 0 30000 Sample Volume [μl] No. Name pH Cr(III)Cl3  8DP_CR(III)_pH7 7 48 16 DP_Cr(III)_pH7.4 7.4 48 24 DP_Cr(III)_pH7.8 7.848

TABLE 12 Antigen recovery determined by SEC-HPLC of exp. 20110913(NIV).For pH 7 and pH 8 the recoveries are based on non-spiked NIV controlsamples #1 and #21, respectively. Samples were stored at 22° C. for 3weeks. Samples marked with “n.a” were not analyzed due to sampleprioritization Results Area Recovery Sample mAU*s (%) 1 NIV_unspiked_pH7_22° C. 4.054 100.0 2 NIV_Ni(II)_100_pH 7_22° C. 3.972 98.0 3NIV_Ni(II)_500_pH 7_22° C. 4.454 109.9 4 NIV_Ni(II)_1000_pH 7_22° C.4.185 103.2 5 NIV_Cu(II)_100_pH 7_22° C. 3.913 96.5 6 NIV_Cu(II)_500_pH7_22° C. 3.733 92.1 7 NIV_Cu(II)_1000_pH 7_22° C. 3.115 76.8 8NIV_CR(III)_100_pH 7_22° C. 3.957 97.6 9 NIV_CR(III)_500_pH 7_22° C.4.000 98.7 10 NIV_CR(III)_1000_pH 7_22° C. 3.611 89.1 11 NIV_PS spike_pH7_22° C. 4.068 100.3 12 NIV_Ni(II)_100_PSspike_pH 7_22° C. 3.884 95.8 13NIV_Ni(II)_500_PSspike_pH 7_22° C. 3.688 91.0 14NIV_Ni(II)_1000_PSspike_pH 7_22° C. 3.971 98.0 15NIV_Cu(II)_100_PSspike_pH 7_22° C. 3.568 88.0 16NIV_Cu(II)_500_PSspike_pH 7_22° C. 3.325 82.0 17NIV_Cu(II)_1000_PSspike_pH 7_22° C. 3.486 86.0 18NIV_CR(III)_100_PSspike_pH 7_22° C. 3.747 92.4 19NIV_CR(III)_500_PSspike_pH 7_22° C. 3.904 96.3 20NIV_CR(III)_1000_PSspike_pH 7_22° C. 3.685 90.9 21 NIV_unspiked_pH 8_22°C. 4.213 100.0 22 NIV_Ni(II)_100_pH 8_22° C. 4.181 99.2 23NIV_Ni(II)_500_pH 8_22° C. 4.150 98.5 24 NIV_Ni(II)_1000_pH 8_22° C.3.772 89.5 25 NIV_Cu(II)_100_pH 8_22° C. 4.146 98.4 26 NIV_Cu(II)_500_pH8_22° C. 4.212 100.0 27 NIV_Cu(II)_1000_pH 8_22° C. 4.152 98.6 28NIV_CR(III)_100_pH 8_22° C. 4.213 100.0 29 NIV_CR(III)_500_pH 8_22° C.3.997 94.9 30 NIV_CR(III)_1000_pH 8_22° C. 4.231 100.4 31 NIV_PSspike_pH 8_22° C. 4.150 98.5 32 NIV_Ni(II)_100_PSspike_pH 8_22° C. 3.62386.0 33 NIV_Ni(II)_500_PSspike_pH 8_22° C. 3.725 88.4 34NIV_Ni(II)_1000_PSspike_pH 8_22° C. 4.079 96.8 35NIV_Cu(II)_100_PSspike_pH 8_22° C. 3.473 82.4 36NIV_Cu(II)_500_PSspike_pH 8_22° C. 3.180 75.5 37NIV_Cu(II)_1000_PSspike_pH 8_22° C. 4.056 96.3 38NIV_CR(III)_100_PSspike_pH 8_22° C. 3.042 72.2 39NIV_CR(III)_500_PSspike_pH 8_22° C. 4.113 97.6 40NIV_CR(III)_1000_PSspike_pH 8_22° C. n.a. n.a.

TABLE 13 ELISA results of exp. 20110913(NIV) obtained after 3 weeks atpH 8 (sample 21-40) and 7 weeks at pH 7(sample 1-20) at 22° C. Samplesmarked with “n.a” were not analyzed due to sample prioritization.20110913_metal spiked_NIV_22° C. 7 weeks at pH 7 No. Name poly monoratio 1 NIV_unspiked_pH7_22° C. CONTROL 20.699 17.512 0.846 2NIV_Ni(II)_100_pH7_22° C. 17.243 14.787 0.858 3 NIV_Ni(II)_500_pH7_22°C. 18.877 16.215 0.859 4 NIV_Ni(II)_1000_pH7_22° C. 16.9 14.522 0.859 5NIV_Cu(II)_100_pH7_22° C. 16.718 13.278 0.794 6 NIV_Cu(II)_500_pH7_22°C. 14.664 5.459 0.372 7 NIV_Cu(II)_1000_pH7_22° C. 7.112 0.421 0.059 8NIV_CR(III)_100_pH7_22° C. 19.207 16.602 0.864 9 NIV_CR(III)_500_pH7_22°C. 20.313 17.84 0.878 10 NIV_CR(III)_1000_pH7_22° C. 16.762 13.128 0.78311 NIV_PS spike_pH7_22° C. CONTROL 19.907 16.994 0.854 12NIV_Ni(II)_100_PSspike_pH7_22° C. 19.546 16.534 0.846 13NIV_Ni(II)_500_PSspike_pH7_22° C. 18.337 15.2 0.829 14NIV_Ni(II)_1000_PSspike_pH7_22° C. 20.759 16.934 0.816 15NIV_Cu(II)_100_PSsPike_pH7_22° C. 18.249 13.348 0.731 16NIV_Cu(II)_500_PSspike_pH7_22° C. 19.201 7.733 0.403 17NIV_Cu(II)_1000_PSspike_pH7_22° C. 8.515 0.711 0.083 18NIV_CR(III)_100_PSspike_pH7_22° C. 18.377 16.521 0.899 19NIV_CR(III)_500_PSspike_pH7_22° C. 19.678 17.119 0.870 20NIV_CR(III)_1000_PSspike_pH7_22° C. 20.505 18.219 0.889 20110913_metalspiked_NIV_22° C. 3 weeks at pH 8 1^(st) analysis 2^(nd) analysis Nopoly mono ratio poly mono ratio 21 NIV_unspiked_pH8_22° C. CONTROL22.729 20.679 0.910 24.687 24.806 1.005 22 NIV_Ni(II)_100_pH8_22° C.23.732 23.572 0.993 24.179 22.148 0.916 23 NIV_Ni(II)_500_pH8_22° C.20.086 19.793 0.985 22.411 22.207 0.991 24 NIV_Ni(II)_1000_pH8_22° C.16.553 15.402 0.930 23.841 21.645 0.908 25 NIV_Cu(II)_100_pH8_22° C.18.736 18.175 0.970 28.024 24.714 0.882 26 NIV_Cu(II)_500_pH8_22° C.21.173 19.109 0.903 25.774 23.983 0.931 27 NIV_Cu(II)_1000_pH8_22° C.19.709 16.406 0.832 24.799 21.580 0.870 28 NIV_CR(III)_100_pH8_22° C.22.464 20.687 0.921 22.782 21.156 0.929 29 NIV_CR(III)_500_pH8_22° C.20.527 20.247 0.986 23.040 21.649 0.940 30 NIV_CR(III)_1000_pH8_22° C.20.838 19.094 0.916 25.676 24.047 0.937 31 NIV_PS spike_pH8_22° C.CONTROL 19.051 20.112 1.056 25.413 25.991 1.023 32NIV_Ni(II)_100_PSspike_pH8_22° C. 18.729 19.250 1.028 n.a. n.a. n.a. 33NIV_Ni(II)_500_PSspike_pH8_22° C. 20.923 20.516 0.981 n.a. n.a. n.a. 34NIV_Ni(II)_1000_PSspike_pH8_22° C. 21.734 19.794 0.911 25.164 24.8990.989 35 NIV_Cu(II)_100_PSspike_pH8_22° C. 21.526 19.852 0.922 n.a. n.a.n.a. 36 NIV_Cu(II)_500_PSspike_pH8_22° C. 21.914 19.449 0.888 n.a. n.a.n.a. 37 NIV_Cu(II)_1000_PSspike_pH8_22° C. 18.646 16.259 0.872 25.37124.275 0.957 38 NIV_CR(III)_100_PSspike_pH8_22° C. 20.292 18.667 0.920n.a. n.a. n.a. 39 NIV_CR(III)_500_PSspike_pH8_22° C. 22.835 21.558 0.94425.561 24.480 0.958 40 NIV_CR(III)_1000_PSspike_pH8_22° C. 27.862 25.2910.908 n.a. n.a. n.a.

TABLE 14 Antigen recoveries after 5 weeks at 22° C. of desorbed JEVobtained by SEC-HPLC. Recoveries were based on non- spiked DP controlsamples stored at either pH 7 or pH 8 JEV area No Sample mAU · minrecovery 1 DP_unspiked_pH 7 22° C. 3.710 100%  2 DP_Ni(II)_100_pH 7 22°C. 3.656 99% 3 DP_Ni(II)_500_pH 7 22° C. 3.705 100%  4 DP_Ni(II)_1000_pH7 22° C. 3.444 93% 5 DP_Cu(II)_100_pH 7 22° C. 3.565 96% 6DP_Cu(II)_500_pH 7 22° C. 3.313 89% 7 DP_Cu(II)_1000_pH 7 22° C. 3.36791% 8 DP_Cr(III)_100_pH 7 22° C. 3.562 96% 9 DP_Cr(III)_500_pH 7 22° C.3.422 92% 10 DP_Cr(III)_1000_pH 7 22° C. 3.148 85% 11 DP_unspiked_pH 822° C. 4.297 100%  12 DP_Ni(II)_100_pH 8 22° C. 4.029 94% 13DP_Ni(II)_500_pH 8 22° C. 4.306 100%  14 DP_Ni(II)_1000_pH 8 22° C.4.065 95% 15 DP_Cu(II)_100_pH 8 22° C. 3.751 87% 16 DP_Cu(II)_500_pH 822° C. 3.698 86% 17 DP_Cu(II)_1000_pH 8 22° C. 3.511 82% 18DP_Cr(III)_100_pH 8 22° C. 3.805 89% 19 DP_Cr(III)_500_pH 8 22° C. 3.84389% 20 DP_Cr(III)_1000_pH 8 22° C. 4.212 98%

TABLE 15 ELISA results of desorbed JEV antigen after 5 weeks at 22° C. %Ratio com- pared to non- Poly. Mono. spiked No. Name pH ELISA ELISARatio Control 1 DP_unspiked_pH7 7 14.203 13.13 0.924 100 2DP_Ni(II)_100_pH7 7 13.089 12.623 0.964 104 3 DP_Ni(II)_500_pH7 7 12.64012.572 0.995 108 4 DP_Ni(II)_1000_pH7 7 15.051 11.757 0.781 84 5DP_Cu(II)_100_pH7 7 13.420 10.792 0.804 87 6 DP_Cu(II)_500_pH7 7 13.24710.079 0.761 82 7 DP_Cu(II)_1000_pH7 7 12.981 9.654 0.744 80 8DP_Cr(III)_100_pH7 7 16.936 11.886 0.702 76 9 DP_Cr(III)_500_pH7 713.991 11.219 0.802 87 10 DP_Cr(III)_1000_pH7 7 13.061 10.438 0.799 8611 DP_unspiked_pH8 8 12.647 11.287 0.892 100 12 DP_Ni(II)_100_pH8 812.308 10.689 0.868 97 13 DP_Ni(II)_500_pH8 8 14.300 12.623 0.883 99 14DP_Ni(II)_1000_pH8 8 12.082 10.930 0.905 101 15 DP_Cu(II)_100_pH8 811.041 9.937 0.900 101 16 DP_Cu(II)_500_pH8 8 9.869 9.176 0.930 104 17DP_Cu(II)_1000_pH8 8 9.379 8.802 0.938 105 18 DP_Cr(III)_100_pH8 810.164 9.545 0.939 105 19 DP_Cr(III)_500_pH8 8 11.241 10.057 0.895 10020 DP_Cr(III)_1000_pH8 8 12.400 11.183 0.902 101

TABLE 16 ELISA results of desorbed JEV antigen after 4 weeks and 7 weeksstored at 22° C. Samples marked with “n.a” were not analyzed due tosample prioritization 4 weeks @ 22° C. 7 weeks @ 22° C. No pH poly monoratio poly mono ratio m/p 1 DP Fe(II) pH 7 7 14.285 11.213 0.785 14.18111.378 0.802 2 DP Fe(III) pH 7 7 14.879 11.552 0.776 14.323 11.765 0.8213 DP Ni(II) pH 7 7 16.572 11.862 0.716 14.231 11.666 0.820 4 DP Co(II)pH 7 7 12.81 12.629 0.986 14.246 11.244 0.789 5 DP Cu(II) pH 7 7 12.4749.747 0.781 11.464 7.654 0.668 6 DP Zn(II) pH 7 7 13.131 11.186 0.85215.122 11.514 0.761 25 DP Cr(III) pH 7 7 12.144 11.598 0.955 13.54310.73 0.792 7 DP metalmix pH 7 7 11.078 8.366 0.755 8.617 5.392 0.626 8DP unspiked pH 7 7 16.159 13.224 0.818 14.158 11.559 0.816 9 DP Fe(II)pH 7.4 7.4 15.096 12.649 0.838 14.417 11.239 0.780 10 DP Fe(III) pH 7.47.4 13.509 12.029 0.890 16.948 12.344 0.728 11 DP Ni(II) pH 7.4 7.413.036 11.306 0.867 13.293 12.571 0.946 12 DP Co(II) pH 7.4 7.4 13.71511.714 0.854 13.079 11.96 0.914 13 DP Cu(II) pH 7.4 7.4 14.235 11.7480.825 10.843 9.276 0.855 14 DP Zn(II) pH 7.4 7.4 13.815 12.882 0.93213.28 12.619 0.950 26 DP Cr(III) pH 7.4 7.4 12.324 11.659 0.946 13.31210.804 0.812 15 DP_metalmix_pH7.4 7.4 n.a. 9.74 6.551 0.673 16 DPunspiked 7.4 13.951 13.225 0.948 11.97 12.672 1.059 pH 7.4 17 DP Fe(II)pH 7.8 7.8 14.356 13.102 0.913 12.625 13.304 1.054 18 DP Fe(III) pH 7.87.8 13.554 12.388 0.914 13.003 10.811 0.831 19 DP Ni(II) pH 7.8 7.813.949 12.496 0.896 13.106 11.757 0.897 20 DP Co(II) pH 7.8 7.8 12.82611.931 0.930 13.333 11.059 0.829 21 DP Cu(II) pH 7.8 7.8 12.593 11.2680.895 12.538 9.872 0.787 22 DPZn(II) pH 7.8 7.8 15.217 14.204 0.93315.55 13.217 0.850 27 DP Cr(III) pH 7.8 7.8 12.977 13.228 1.019 14.64211.975 0.818 23 DP metalmix pH 7.8 7.8 11.196 9.811 0.876 10.771 7.5390.700 24 DP unspiked 7.8 11.906 11.819 0.993 12.472 11.034 0.885 pH 7.8

TABLE 17 ANOVA for stability samples stored at 22° C. for 7 weeks.Analysis of Variance for Ratio - Type III Sums of Squares Sum of Mean F-P- Source Squares Df Square Ratio Value MAIN EFFECTS A: Metal Type0.139643 8 0.0174554 3.00 0.0293 B: pH 0.0462028 2 0.0231014 3.97 0.0398RESIDUAL 0.0931046 16 0.00581904 TOTAL (CORRECTED) 0.27895 26 AllF-ratios are based on the residual mean square error.

TABLE 18 Multiple range test for ratio by metal ion type. 1 = Fe(II); 2= Fe(III); 3 = Ni(II); 4 = Co(II); 5 = Cu(II); 6 = Zn(II); 7 = Cr(III);8 = Mix[1-6]; 9 = non-spiked control Multiple Range Tests for Ratio byMetal Type Method: 95.0 percent LSD Homogeneous Metal Type Count LS MeanGroups 8 3 0.666087 X 5 3 0.770168 XX 2 3 0.793725 XXX 7 3 0.807248 XX 43 0.844388 XX 6 3 0.853867 XX 1 3 0.878563 XX 3 3 0.887505 XX 9 30.919926 X Contrast Difference +/−Limits 1 − 2 0.084838 0.132037 1 − 3−0.0089421 0.132037 1 − 4 0.0341754 0.132037 1 − 5 0.108395 0.132037 1 −6 0.0246961 0.132037 1 − 7 0.0713155 0.132037 1 − 8 *0.212476 0.132037 1− 9 −0.0413627 0.132037 2 − 3 −0.0937801 0.132037 2 − 4 −0.05066260.132037 2 − 5 0.0235569 0.132037 2 − 6 −0.0601419 0.132037 2 − 7−0.0135225 0.132037 2 − 8 0.127638 0.132037 2 − 9 −0.126201 0.132037 3 −4 0.0431175 0.132037 3 − 5 0.117337 0.132037 3 − 6 0.0336382 0.132037 3− 7 0.0802576 0.132037 3 − 8 *0.221418 0.132037 3 − 9 −0.03242060.132037 4 − 5 0.0742195 0.132037 4 − 6 −0.00947935 0.132037 4 − 70.0371401 0.132037 4 − 8 *0.1783 0.132037 4 − 9 −0.0755381 0.132037 5 −6 −0.0836988 0.132037 5 − 7 −0.0370794 0.132037 5 − 8 0.104081 0.1320375 − 9 *−0.149758 0.132037 6 − 7 0.0466195 0.132037 6 − 8 *0.187780.132037 6 − 9 −0.0660588 0.132037 7 − 8 *0.14116 0.132037 7 − 9−0.112678 0.132037 8 − 9 *−0.253838 0.132037 *denotes a statisticallysignificant difference.

TABLE 19 ICP-MS results of residual metal ion impurities present invarious Alum (2%) lots Residual metal content (ng/mL) Alum (2%) Lot CrFe Ni Cu V Co 4074 19.8 266 14.8 <25 <5 <5 4470 1637 1179 17.5 <25 <5 <54563 1874 2485 8.9 <25 <5 <5 4621 1333 1183 7.6 <25 <5 <5 3877 48.2 18312.2 <25 <5 <5 4230 (nonGI**, 1139 5640 816 64 12.6 7 GI***) Mix*4074/4230 579.4 2953 415.4 <44.5 <6 <6 *Calculated content of residualmetals based on Alum lot 4230 and 4074 **nonGI: non gamma irradiated***GI: gamma irradiated

TABLE 20 Summary of metal ion content and analysis of DP samplesformulated with various Alum lots. Samples were analysed in duplicate byELISA and the ratio of monoclonal/polyclonal ELISA is reported.Formulations were stored at 22° C. for 6 weeks. Alum Lot (2% StockMonoclonal Polyclonal Monoclonal Polyclonal Ratio Ratio Mean ratioRange* # solution) 1^(st) analysis 1^(st) analysis 2^(nd) analysis2^(nd) analysis 1^(st) analysis 2^(nd) analysis (Mono/poly) Ratio 1 447023.584 26.397 21.666 22.948 0.893 0.944 0.918 0.025 2 4563 23.027 23.39719.051 18.862 0.984 1.010 0.997 0.013 3 4621 22.758 24.056 19.041 19.1960.946 0.991 0.968 0.023 4 3877 23.5 23.85 21.186 21.24 0.985 0.997 0.9910.006 5 4230 21.85 25.155 20.682 23.792 0.868 0.869 0.869 0.000(non-gamma irradiated) 6 4230 20.509 22.904 18.002 23.512 0.895 0.7650.830 0.065 (gamma irradiated) 7 4074 23.022 24.047 16.695 19.866 0.9570.840 0.898 0.058 8 Mixture 20.833 22.954 22.473 21.217 0.908 1.0590.983 0.076 (50%/50%) of 4074 and 4230 *Range is the absolute differencebetween 1^(st) and 2^(nd) analysis.

TABLE 21 Results of PSD analysis of ALHYDROGEL ® (2%) stock solution inwater. Obscuration No Sample Name d (0.1) d (0.5) d (0.9) (%) 1Non-irradiated AlOH 0.70 2.13 46.53 1.51 RQCS0890 Lot 4230 2 GI AlOHRQCS1200 0.71 4.14 69.64 1.98 Lot 4230 3 GI AlOH RQCS1342 0.78 2.2353.44 2.11 Lot 4740 4 GI AlOH RQCS0448 0.73 4.49 78.58 1.96 Lot 4074 d(0.1): 10% of all measured particles have a diameter below this value d(0.5): 50% of all measured particles have a diameter below this value d(0.9): 90% of all measured particles have a diameter below this valueObscuration: amount of laser light reduction by sample; corresponds toconcentration of sample in measurement chamber

TABLE 22 Results of ALHYDROGEL ® titration curves for determination ofPOZ. Samples were analyzed in PBS (1:20 dilution). Sample ID PZC (pH)Non-irradiated AlOH 4.58 RQCS0890 Lot 4230 GI AlOH RQCS1200 4.62 Lot4230 GI AlOH RQCS1342 4.49 Lot 4740 GI AlOH RQCS0448 4.48 Lot 4074

TABLE 23 Summary of metal ion analysis for various Aluminum hydroxidestock solutions; Note: A 2% stock solution equals 10 mg/mL of AlALHYDROGEL ® (2% Al Fe Ni Cu Co Cr Ag Cd W Pb V Rb Mo solution) μg/mLng/mL Lot 4074 (RQCS0013) 9130 266 15 <25 <5 20 <5 <5 <25 19 <5 <5 <5Lot 4230 (RQCS 0890) 9570 5640 816 64 7 1139 <5 <5 <25 24 13 <5 11 Lot4470 (RQCS1254) 9560 1179 18 <25 <5 1637 <5 <5 <25 20 <5 <5 22 Lot 4414(RQCS1220) 10272 2790 36 <25 <5 1710 <5 <5 35 <25  <7 n.a. n.a. Lot 4539n.a. 943 119 <25 <5 276 <5 <5 <25 27 <5 <5 <5 Lot 3877 10766 183 12 <25<5 48 <5 <5 <25 10 <5 <5 <5 Lot 4187 14100 3617 172 <25 <5 2333 <5 <5<25 18 <5 <5 <5 Lot 4287 9800 2047 296 <25 <5 620 <5 <5 <25 30 10 <5 <5Lot 4563 9360 2485 9 <25 <5 1874 <5 <5 <25  8 <5 <5 <5 Lot 4621 97601183 8 <25 <5 1333 <5 <5 <25  8 <5 <5 <5 Lot 4580 (7xwashed) 10497 261027 <25 <5 1470 <5 <5 <25 <25  <5 n.a. n.a. Lot 4596 (7xwashed) 107763530 27 <25 <5 1710 <5 <5 <25 <25  <5 n.a. n.a. Lot 4577 (7xwashed)10720 3060 24 <25 <5 1650 <5 <5 <25 <25  <5 n.a. n.a. average 10359 2272121 n.a.** <5 1217 <5 <5 n.a.**  18*  11* <5  16* stdev 1312 1538 225745  8  2  8 min 9130 183 8 <25 <5 20 <5 <5 <25  8 10 <5 <5 max 141005640 816 64 7 2333 <5 <5 35 30 13 <5 22 RSD (%) 13 68 186 61 44 14 47*results below LOQ were not used for average calculation; **no averagecalculation possible due to results below LOQ

TABLE 24 Analysis of supernatant and Aluminum hydroxide (Lot 4230) gelfraction for contaminating metal ions shows metal ions are located inthe gel, not the supernatant Fe Ni Cu Co Cr Ag Cd W Pb V Sample ng/mLLot 4230 Supernatant 82 12 <25 <5 7 <5 <5 <25 70 <5 Lot 4230 Sediment6200 920 <25 <5 1200 <5 <5 <25 45 13 % supernatant 1.3 1.3 n.a. n.a. 0.6n.a. n.a. n.a. 155.6 n.a. compared to sediment

Preferred Aspects

Aspect 1. A method for preparing an aqueous composition comprisingaluminium and a protein said method comprising

-   -   combining an aluminium-salt, said protein and water to produce        said aqueous composition and    -   determining the level of a heavy metal in the aqueous        composition and/or the aluminium-salt.

Aspect 2. A method for preparing an aqueous composition comprisingaluminium and a protein said method comprising

-   -   preparing or selecting an aluminium-salt that is able to provide        an aqueous composition having less than 350 ppb heavy metal        based on the weight of the aqueous composition and    -   combining said aluminium salt, said protein and water to produce        said aqueous composition.

Aspect 3. A method according to aspect 1-2, further comprising bufferingsaid aqueous composition at a pH of between 6.5 and 8.5, preferably at apH between 7.5 and 8.5.

Aspect 4. A method according to aspect 1-3, further comprising packagingaliquots of said aqueous composition having less than 350 ppb heavymetal based on the weight of the aqueous composition in separateair-tight storage containers.

Aspect 5. A method for preparing a clinical grade aluminium-saltprecipitate for incorporation into a medicament and/or vaccine, saidmethod comprising preparing an aqueous solution of aluminium ions andprecipitating said aluminium-ions from said solution, and determiningthe level of a heavy metal in the solution and/or the aluminium-saltprecipitate.

Aspect 6. A method according to aspect 5, wherein the precipitate isselected that is able to provide an aqueous composition comprising lessthan 350 ppb heavy metal based on the weight of the aqueous composition.

Aspect 7. An aqueous composition comprising a protein and analuminium-salt, said composition comprising less than 350 ppb heavymetal based on the weight of the aqueous composition.

Aspect 8. An aqueous composition according to aspect 7, which has beenstored at temperatures higher than 20° C. for at least 1 month.

Aspect 9. An aqueous composition according to aspect 7 having ashelf-life of at least 20 month.

Aspect 10. An aqueous composition according to aspect 7-9, wherein saidheavy metal is selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr,Pb, Rb and Mo.

Aspect 11. An aqueous composition according to aspect 7-10, wherein saidheavy metal is selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V.

Aspect 12. An aqueous composition according to aspect 7-11, wherein saidheavy metal is selected from Cu or Ni.

Aspect 13. An aqueous composition according to aspect 7-12, wherein saidheavy metal is present in ionic form.

Aspect 14. An aqueous composition according to aspect 7-13, wherein thealuminium-salt is aluminiumhydroxide (Al(OH)3) or aluminiumphosphate(AlPO4).

Aspect 15. An aqueous composition according to aspect 7-14, furthercomprising a reactive compound.

Aspect 16. An aqueous composition according to aspect 15, wherein thereactive compound is selected from the group consisting of a redoxactive compound, a radical building compound, a stabilizing compound anda combination of any thereof.

Aspect 17. An aqueous composition according to aspect 15-16, wherein thereactive compound is selected from the group consisting of formaldehyde,ethanol, chloroform, trichloroethylene, acetone, TRITON™ X-100(Polyethylene glycol tert-octylphenyl ether), deoxycholate,diethylpyrocarbonate, sulphite, Na₂S₂O₅, beta-proprio-lacton,polysorbate such as TWEEN® 20 (Polysorbate 20) or TWEEN® 80 (Polysorbate80), O₂, phenol, PLURONIC (poloxamer) type copolymers, and a combinationof any thereof.

Aspect 18. An aqueous composition according to aspect 7-17, comprisingbetween 5 μg/ml and 50 mg/ml aluminium.

Aspect 19. An aqueous composition according to aspect 7-18, comprisingbetween 50 μg/ml and 5 mg/ml aluminium.

Aspect 20. An aqueous composition according to aspect 7-19, comprisingbetween 5 ppb and 250 ppb Fe based on the weight of the aqueouscomposition.

Aspect 21. An aqueous composition according to aspect 7-20, comprisingless than 3 ppb Cu based on the weight of the aqueous composition.

Aspect 22. An aqueous composition according to aspect 7-21, comprisingless than 40 ppb Ni based on the weight of the aqueous composition.

Aspect 23. An aqueous composition according to aspect 7-22, wherein saidprotein is a therapeutic and/or a vaccine.

Aspect 24. An aqueous composition according to aspect 7-23, wherein saidprotein is a viral or bacterial protein.

Aspect 25. An aqueous composition according to aspect 7-24, wherein saidviral protein is a protein of the Japanese encephalitis virus or aprotein of the Pseudomonas aeruginosa bacterium.

Aspect 26. An aqueous composition according to aspect 7-25, wherein saidprotein is protein within a formaldehyde inactivated virus particles.

Aspect 27. An aqueous composition according to aspect 7-26, furthercomprising sulphite.

Aspect 28. An aqueous composition according to aspect 7-27, obtained bya method according to aspect 1-6.

Aspect 29. A vaccine comprising an aqueous composition according toaspect 7-28.

Aspect 30. An aluminium hydroxide concentrate that a) comprises 10 mg/mlof said aluminium hydroxide and b) less than 7 microgram heavy metal,for use in the manufacture of a medicine, preferably for use in themanufacture of a vaccine.

Aspect 31. An aluminium hydroxide concentrate that comprises 10 lessthan 7 microgram heavy metal, for use in the manufacture of a medicine,preferably for use in the manufacture of a vaccine.

Aspect 32. An aluminium salt concentrate that comprises less than 7 ppmheavy metal based on the weight of the concentrate, for use in themanufacture of a medicine, preferably for use in the manufacture of avaccine.

Aspect 33. An aluminium salt concentrate that comprises less than 700ppm heavy metal based on the weight of the aluminium salt, for use inthe manufacture of a medicine, preferably for use in the manufacture ofa vaccine.

Aspect 34. An aluminium hydroxide concentrate that comprises less than700 ppm heavy metal based on the weight of the aluminium hydroxide, foruse in the manufacture of a medicine, preferably for use in themanufacture of a vaccine.

Aspect 35. A method for preparing an aqueous composition comprisingaluminium, a reactive compound and a protein said method comprising

-   -   preparing or selecting an aluminium-salt that is able to provide        an aqueous composition having less than 350 ppb heavy metal        based on the weight of the aqueous composition and    -   combining said aluminium salt, said protein, reactive compound        and water to produce said aqueous composition.

Aspect 36. The method for preparing an aqueous composition according toaspect 35, wherein said heavy metal is selected from Cu, Ni, W, Co, Os,Ru, Cd, Ag, Fe, V, Cr, Pb, Rb and Mo.

Aspect 37. The method for preparing an aqueous composition according toaspect 35-36, wherein said heavy metal is selected from Cu, Ni, W, Co,Os, Ru, Cd, Ag, Fe, V.

Aspect 38. The method for preparing an aqueous composition according toaspect 35-37, wherein said heavy metal is selected from Cu or Ni.

Aspect 39. The method for preparing an aqueous composition according toaspect 35-38, wherein said heavy metal is present in ionic form.

Aspect 40. The method for preparing an aqueous composition according toaspect 35-39, wherein the aluminium-salt is aluminiumhydroxide (Al(OH)3)or aluminiumphosphate (AlPO4).

Aspect 41. The method for preparing an aqueous composition according toaspect 35-40, wherein the reactive compound is selected from the groupconsisting of a redox active compound, a radical building compound, astabilizing compound and a combination of any thereof.

Aspect 42. The method for preparing an aqueous composition according toaspect 35-41, wherein the reactive compound is selected from the groupconsisting of formaldehyde, ethanol, chloroform, trichloroethylene,acetone, TRITON™ X-100 (Polyethylene glycol tert-octylphenyl ether),deoxycholate, diethylpyrocarbonate, sulphite, Na₂S₂O₅,beta-proprio-lacton, polysorbate such as TWEEN® 20 (Polysorbate 20) orTWEEN® 80 (Polysorbate 80), O₂, phenol, PLURONIC (poloxamer) typecopolymers, and a combination of any thereof.

Aspect 43. The method for preparing an aqueous composition according toaspect 35-42, comprising between 5 μg/ml and 50 mg/ml aluminium.

Aspect 44. The method for preparing an aqueous composition according toaspect 35-43, comprising between 50 μg/ml and 5 mg/ml aluminium.

Aspect 45. The method for preparing an aqueous composition according toaspect 35-44, comprising between 5 ppb and 250 ppb Fe based on theweight of the aqueous composition.

Aspect 46. The method for preparing an aqueous composition according toaspect 35-45, comprising less than 3 ppb Cu based on the weight of theaqueous composition.

Aspect 47. The method for preparing an aqueous composition according toaspect 35-46, comprising less than 40 ppb Ni based on the weight of theaqueous composition.

Aspect 48. A method for preparing an aqueous composition according toaspect 35-47, further comprising buffering said aqueous composition at apH of between 6.5 and 8.5, preferably 7.5 and 8.5.

Aspect 49. A method for preparing an aqueous composition according toaspect 35-48, further comprising packaging aliquots of said aqueouscomposition in separate air-tight storage containers.

Aspect 50. A method for prevention and/or treatment of a subject in needthereof that comprises the administration of an aqueous compositioncomprising an effective dose of an antigen, an aluminium compound, areactive compound and less than 350 ppb heavy metal based on the weightof the aqueous composition.

Aspect 51. A method for prevention and/or treatment of a subject in needthereof that comprises the administration of an effective dose of acomposition according to aspect 7-29.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety for the purposes cited herein.

What is claimed is:
 1. An aqueous composition comprising a protein andan aluminum-salt, said composition comprising less than 1.25 ppb Cubased on the weight of the aqueous composition, and wherein said proteinis protein within formaldehyde inactivated virus particles.
 2. Anaqueous composition according to claim 1, which has been stored attemperatures higher than 20° C. for at least 1 month.
 3. An aqueouscomposition according to claim 1, comprising less than 350 ppb heavymetal, wherein said heavy metal is selected from Ni, W, Co, Os, Ru, Cd,Ag, Fe, V, Cr, Pb, Rb and Mo.
 4. An aqueous composition according toclaim 1, comprising less than 350 ppb heavy metal, wherein said heavymetal is selected from Ni, W, Co, Os, Ru, Cd, Ag, Fe, V.
 5. An aqueouscomposition according to claim 1, comprising less than 350 ppb heavymetal, wherein said heavy metal is selected from Ni.
 6. An aqueouscomposition according to claim 1, comprising less than 350 ppb heavymetal, wherein said heavy metal is present in ionic form.
 7. An aqueouscomposition according to claim 1, wherein the aluminum-salt is aluminumhydroxide (Al(OH)3) or aluminum phosphate (AlPO4).
 8. An aqueouscomposition according to claim 1, wherein the aluminum-salt is aluminumhydroxide (Al(OH)3).
 9. An aqueous composition according to claim 1,further comprising a reactive compound selected from the groupconsisting of a redox active compound, a radical building compound, astabilizing compound and a combination of any thereof.
 10. An aqueouscomposition according to claim 1, further comprising a reactive compoundselected from the group consisting of formaldehyde, ethanol, chloroform,trichloroethylene, acetone, polyethylene glycol tert-octylphenyl ether,deoxycholate, diethylpyrocarbonate, sulphite, Na₂S₂O₅,beta-proprio-lacton, polysorbate optionally Polysorbate 20 orPolysorbate 80, O₂, phenol, pluronic type copolymers, and a combinationof any thereof.
 11. An aqueous composition according to claim 1,comprising between 5 μg/ml and 50 mg/ml aluminum.
 12. An aqueouscomposition according to claim 1, comprising between 50 μg/ml and 5mg/ml aluminum.
 13. An aqueous composition according to claim 1,comprising between 5 ppb and 250 ppb Fe based on the weight of theaqueous composition.
 14. An aqueous composition according to claim 1,comprising less than 40 ppb Ni based on the weight of the aqueouscomposition.
 15. An aqueous composition according to claim 1, whereinsaid protein is a therapeutic and/or a vaccine.
 16. An aqueouscomposition according to claim 1, further comprising sulphite.
 17. Avaccine comprising an aqueous composition according to claim 1.